Expression ofStreptomyces melC andchoA genes by a clonedStreptococcus thermophilus promoter STP2201 |
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Authors: | D K Y Solaiman G A Somkuti |
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Affiliation: | (1) Agricultural Research Service, Eastern Regional Research Center, US Department of Agriculture, 600 East Mermaid Lane, 19118 Philadelphia, PA, USA |
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Abstract: | Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected inEscherichia 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 inE. coli. Subcloning of a STP2201-melC DNA fragment into the pMEU-seriesS. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel+ phenotype toE. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase production (2 units mg–1 protein) inE. coli, and to produce an apparently inactivemelC gene product that reacts with anti-tyrosinase antiserum inS. thermophilus. SubstitutingmelC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to anE. coli transformant at a level of (1.06±0.15)×10–7 units mg–1 protein. Introduction of this plasmid intoS. thermophilus by electrotransformation yielded ChoA transformant that produced the enzyme at about 25% of the level found inE. coli. |
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Keywords: | lactic acid bacteria promoter melanin tyrosinase cholesterol oxidase |
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