Abstract
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 Plac-mediated cho expression in a pUCO193-containing host. The recombinant pUCO195P-36 appeared to be segregationally less stable in E. coli DH5α 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) × 10−3 U/mg and (16.0±1.0) × 10−3 U/mg protein equivalent of cell extract in the absence and presence of isopropyl β-d-thio-galactopyranoside, respectively. The presence of a counter-oriented Plac at the 3′ end of the operon in the pMCU22bPa plasmid reduced the sP1-mediated tyrosinase production by about 85%.
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References
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1987) Current protocols in molecular biology. Wiley-Interscience, New York
Bernan V, Filpula D, Herber W, Bibb M, Katz E (1985) The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product. Gene 37:101–110
Betancourt AM, Bernan V, Herber W, Katz E (1992) Analysis of tyrosinase synthesis in Streptomyces antibioticus. J Gen Microbiol 138:787–794
Bojovic B, Djordjevic G, Topisirovic L (1991) Improved vector for promoter screening in lactococci. Appl Environ Microbiol 57:385–388
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
della-Cioppa G, Garger SJ, Sverlow GG, Turpen TH, Grill LK (1990) Melanin production in Escherichia coli from a cloned tyrosinase gene. Biotechnology 8:634–638
Forster AC, McInnes JL, Skingle DC, Symons RH (1985) Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent photobiotin. Nucleic Acid Res 13:745–761
Hintermann G, Zatchej M, Hutter R (1985) Cloning and expression of the genetically unstable tyrosinase structural gene from Streptomyces glaucescens. Mol Gen Genet 200:422–432
Horii M, Ishizaki T, Paik S-Y, Manome T, Murooka Y (1990) An operon containing the genes for cholesterol oxidase and a cytochrome P-450-like protein from a Streptomyces sp. J Bacteriol 172:3644–3653
Johnson TL, Somkuti GA (1990) Properties of cholesterol dissimilation by Rhodococcus equi. J Food Protect 53:332–335
Katz E, Thompson CJ, Hopwood DA (1983) Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol 129:2703–2714
Koivula T, Sibakov M, Palva I (1991) Isolation and characterization of Lactococcus lactis subsp. lactis promoters. Appl Environ Microbiol 57:333–340
Lerch K, Ettlinger L (1972) Purification of a tyrosinase from Streptomyces glaucescens. Eur J Biochem 31:427–437
Leu W-M, Wu S-Y, Lin J-J, Lo SJ, Lee Y-HW (1989) Analysis of the promoter region of the melanin locus from Streptomyces antibioticus. Gene 84:267–277
Lisser S, Margalit H (1993) Compilation of E. coli mRNA promoter sequences. Nucleic Acids Res 21:1507–1516
Murooka Y, Ishizaki T, Nimi O, Maekawa N (1986) Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702. Appl Environ Microbiol 52:1382–1385
Rodriguez-Lopez JN, Escribano J, Garcia-Canovas F (1994) A continuous spectrophotometric method for the determination of monophenolase activity of tyrosinase using 3-methyl-2-benzothiazolinone hydrazone. Anal Biochem 216:205–212
Slos P, Bourquin J-C, Lemoine Y, Mercenier A (1991) Isolation and characterization of chromosomal promoters of Streptococcus salivarius subsp. thermophilus. Appl Environ Microbiol 57:1333–1339
Solaiman DKY, Somkuti GA (1991) Expression of streptomycete cholesterol oxidase in Escherichia coli. J Ind Microbiol 8:253–258
Solaiman DKY, Somkuti GA (1992) DNA structures contributing to the instability of recombinant plasmids in Streptococcus thermophilus. Biotechnol Lett 14:753–758
Solaiman DKY, Somkuti GA (1993) Shuttle vectors developed from Streptococcus thermophilus native plasmid. Plasmid 30:67–78
Solaiman DKY, Somkuti GA, Steinberg DH (1992) Construction and characterization of shuttle plasmids for lactic acid bacteria and Escherichia coli. Plasmid 28:25–36
Somkuti GA, Solaiman DKY, Johnson TL, Steinberg DH (1991) Transfer and expression of a Streptomyces cholesterol oxidase gene in Streptococcus thermophilus. Biotechnol Appl Biochem 13:238–245
Somkuti GA, Solaiman DKY, Steinberg DH (1992) Expression of Streptomyces sp. cholesterol oxidase in Lactobacillus casei. Appl. Microbiol Biotechnol 37:330–334
Sugiyama M, Nomura H, Nimi O (1990) Use of the tyrosinase gene from Streptomyces to probe promoter sequences for Escherichia coli. Plasmid 23:237–241
Tseng H-C, Lin C-K, Hsu B-J, Leu W-M, Lee Y-HW, Chiou S-J, Hu N-T, Chen CW (1990) The melanin operon of Streptomyces antibioticus: expression and use as a marker in gram-negative bacteria. Gene 86:123–128
Vossen JMBM van der, Lelie D van der, Venema G (1987) Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol 53:2452–2457
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Solaiman, D.K.Y., Somkuti, G.A. Expression of cho and melC operons by a Streptococcus thermophilus synthetic promoter in Escherichia coli . Appl Microbiol Biotechnol 43, 285–290 (1995). https://doi.org/10.1007/BF00172826
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DOI: https://doi.org/10.1007/BF00172826