Expression of cho and melC operons by a Streptococcus thermophilus synthetic promoter in Escherichia coli.
A 63-base-pair synthetic promoter, sP1, was synthesized on the basis of the nucleotide sequence of a putative Streptococcus thermophilus promoter. When inserted upstream from the Streptomyces cho operon in a recombinant plasmid, pUCO195P-36, sP1 activated the expression of the cho genes in Escherichia coli, as shown by the production of cholesterol oxidase by the transformants. The sP1-driven cholesterol oxidase production in pUCO195P-36-transformed cells was estimated to be 40% of that produced by P(lac)-mediated cho expression in a pUCO193-containing host. The recombinant pUCO195P-36 appeared to be segregationally less stable in E. coli DH5 alpha than in HB101. Its non-expressing counterpart, pUCO195P-1, was stable in both E. coli strains. The activity of sP1 was further demonstrated in E. coli by the expression of a Streptomyces melC operon. When placed upstream from the test operon in the pMCU22aPa construct, sP1 activated the melC expression as shown by the production of tyrosinase at (3.0 +/- 0.3) x 10(-3) U/mg and (16.0 +/- 1.0) x 10(-3) U/mg protein equivalent of cell extract in the absence and presence of isopropyl beta-D-thiogalactopyranoside, respectively. The presence of a counter-oriented P(lac) at the 3' end of the operon in the pMCU22bPa plasmid reduced the sP1-mediated tyrosinase production by about 85%.
Solaiman
D K
DK
U.S. Department of Agriculture, Agricultural Research Service, Philadelphia, PA 19118, USA.
Somkuti
G A
GA
D001483:Base Sequence; D002789:Cholesterol Oxidase / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005813:Genes, Synthetic / Q000235:genetics*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000235:genetics; D009876:Operon; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013291:Streptococcus / Q000235:genetics*
D002789:Cholesterol Oxidase; D014442:Monophenol Monooxygenase