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 o f the nucleotide sequence of a putative Streptococcus thermophilus pro moter. When inserted upstream from the Streptomyces cho operon in a re combinant plasmid, pUCO195P-36, sP1 activated the expression of the ch o genes in Escherichia coli, as shown by the production of cholesterol oxidase by the transformants. The sP1-driven cholesterol oxidase prod uction in pUCO195P-36-transformed cells was estimated to be 40% of tha t produced by P-lac-mediated cho expression in a pUCO193-containing ho st. The recombinant pUCO195P-36 appeared to be segregationally less st able in E. coli DH5 alpha than in HB101. Its nonexpressing 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 Streptomyce s melC operon. When placed upstream from the test operon in the pMCU22 aPa construct, sP1 activated the melC expression as shown by the produ ction 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 pres ence of isopropyl beta-D-thiogalactopyranoside, respectively. The pres ence of a counter-oriented P-lac at the 3' end of the operon in the pM CU22bPa plasmid reduced the sP1-mediated tyrosinase production by abou t 85%.