EXPRESSION OF STREPTOMYCES MELC AND CHOA GENES BY A CLONED STREPTOCOCCUS-THERMOPHILUS PROMOTER STP2201

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments ha ving promoter activity were cloned and selected in Escherichia coli us ing a chloramphenicol acetyltransferase-(cat-) based promoter-probe ve ctor pKK520-3, Insertion of a promoterless streptomycete melanin biosy nthesis operon (melC) downstream from the promoters of the library fur ther identified clone STP2201 as a strong promoter in E. coli, Subclon ing of a STP2201-melC DNA fragment into the pMEU-series S. thermophilu s - E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferr ed Mel(+) phenotype to E. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase production (2 units mg(-1) protein) in E. coli, and to produce an apparently inactive melC gene product th at reacts with anti-tyrosinase antiserum in S. thermophilus. Substitut ing melC with a streptomycete cholesterol oxidase gene (choA) in the s ame orientation yielded pEU5aCH2201a that conferred ChoA activity to a n E. coli transformant at a level of (1.06 +/- 0.15) x 10(-7) units mg (-1) protein, Introduction of this plasmid into S. thermophilus by ele ctrotransformation yielded ChoA(+) transformant that produced the enzy me at about 25% of the level found in E. coli.