Expression of Streptomyces melC and choA genes by a cloned Streptococcus thermophilus promoter STP2201.
Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected in Escherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone STP2201 as a strong promoter in E. coli. Subcloning of a STP2201-melC DNA fragment into the pMEU-series S. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel+ phenotype to E. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase pro-anti-tyrosinase antiserum in S. thermophilus. Substituting melC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to an 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 electrotransformation yielded ChoA+ transformant that produced the enzyme at about 25% of the level found in E. coli.
Solaiman
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DK
US Department of Agriculture, Eastern Regional Research Center, Philadelphia, PA 19118, USA.
Somkuti
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GA
D002789:Cholesterol Oxidase / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008543:Melanins / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013291:Streptococcus / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics*
D008543:Melanins; D002789:Cholesterol Oxidase