Summary
The gene for a β-glucosidase from the extremely thermophilic bacterium Caldocellum saccharolyticum has been isolated from a genomic library and sequenced. An open reading frame identified by computer analysis of the sequence could encode a protein of Mr 54400, which is close to the size of the polypeptide experimentally determined using maxicells. Analysis of the amino-terminal residues of the protein produced in Escherichia coli suggests that it is processed by a methionine aminopeptidase. A sequence within C. saccharolyticum DNA upstream of the β-glucosidase gene was found to act as a promoter for expression of the thermophile gene in E. coli. The protein has been overproduced in E. coli and Bacillus subtilis where it retains its enzymatic activity and heat stability. There appears to be a single copy of the gene in Caldocellum DNA.
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References
Béguin P, Cornet P, Aubert J-P (1985) Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum. J Bacteriol 162:102–105
Béguin P, Millet J, Aubert JP (1987) The cloned cel (cellulase degradation) genes of Clostridium thermocellum and their products. Microbiol Sci 4:277–280
Ben-Bassat A, Bauer K (1987) Amino-terminal processing of proteins. Nature 326:315
Bergquist PL, Love DR, Croft JE, Streiff MB, Daniel RM, Morgan WH (1987) Molecular genetics and the biotechnological applications of thermophilic bacteria. Biotechnol Genet Eng Rev 5:199–244
Boer HA de, Comstock LJ, Vasser M (1983) The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci USA 80:21–25
Botstein D, Shortle D (1985) Strategies and applications of in vitro mutagenesis. Science 229:1193–1201
Brendel V, Trifonov EN (1984) A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res 12:4411–4427
Dale RMK, McClure BA, Houchins JP (1985) A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing. Plasmid 13:31–40
Daniel RM (1986) The stability of proteins from extreme thermophiles. In: Oxender DI (ed) Protein structure, folding and design. Genex-UCLA Symposium, Liss, New York, pp 291–296
Donnison AM, Brocklesbury CM, Morgan WH (1986) A new species of non-sporulating thermophilic cellulolytic bacterium. Proc Int Congress Microbiol, Manchester, p 203
Gribskov M, Burgess RR, Devereux J (1986) PEPPLOT, a protein secondary structure analysis program for the UWGCG sequence analysis software package. Nucleic Acids Res 14:327–334
Hohn B, Collins J (1980) A small cosmid for the efficient cloning of large DNA fragments. Gene 11:291–298
Joliff G, Béguin P, Aubert J-P (1986) Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum. Nucleic Acids Res 14:8605–8613
Kohchi C, Toh-e A (1987) Nucleotide sequence of Candida pelliculosa β-glucosidase gene. Nucleic Acids Res 13:6273–6282
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685
Love DR, Streiff MB (1987) Molecular cloning of a β-glucosidase gene from an extremely thermophilic anaerobe in E. coli and B. subtilis. Biotechnology 5:384–387
Matsumara M, Kataoka S, Aiba S (1986) Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase. Mol Gen Genet 204:355–358
Matthews BW, Nicholson H, Bechtel WJ (1987) Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci USA 84:6663–6667
McKenney K, Shimitake H, Court D, Schmeissner U, Brady C, Rosenberg M (1981) A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. In: Chirikjian J, Papas T (eds) Gene amplification and analysis, vol 2. Structural analysis of nucleic acids. Elsevier, New York, pp 383–415
Mulligan ME, Hawley DK, Entriken R, McClure WR (1984) Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res 12:789–800
Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106
Oakley BR, Kirsch DR, Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 105:361–363
Oshima T (1986) The genes and genetic apparatus of extreme thermophiles. In: Brock TD (ed) Thermophiles: general, molecular and applied microbiology. John Wiley and Sons, pp 137–147
Perutz M (1978) Electrostatic effects in proteins. Science 201:1187–1191
Raynal A, Gerbaud C, Francingues MC, Guerineau M (1987) Sequence and transcription of the β-glucosidase gene of Kluyveromyces fragilis cloned in Saccharomyces cerevisiae. Curr Genet 12:175–184
Reed KC, Mann DA (1985) Rapid transfer of DNA from agarose gels to uylon membranes. Nucleic Acids Res 13:7207–7221
Romaniec MPM, Davidson K, Hazlewood GP (1987a) Cloning and expression in Escherichia coli of Clostridium thermocellum DNA encoding β-glucosidase activity. Enzyme Microbiol Technol 9:474–478
Romaniec MPM, Clarke NG Hazlewood GP (1987b) Molecular cloning of Clostridium thermocellum DNA and the expression of further novel endo-β-1,4-glucanase genes in Escherichia coli. J Gen Microbiol 133:1297–1307
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sekiguchi T, Ortega-Cesena J, Nosoh Y, Ohashi S, Tsuda K, Kanaya S (1986) DNA and amino-acid sequences of 3-isopropylmalate dehydrogenase of Bacillus coagulans. Comparison with the enzymes of Saccharomyces cerevisiae and Thermus thermophilus. Biochim Biophys Acta 867:36–44
Williams DM, Duvall EJ, Lovett PS (1981a) Cloning restriction fragments that promote expression of a gene in Bacillus subtilis. J Bacteriol 146:1162–1165
Williams DM, Schoner RG, Duvall EJ, Preis LH, Lovett PS (1981b) Expression of Escherichia coli trp genes and the mouse dihydrofolate reductase gene cloned in Bacillus subtilis. Gene 16:199–206
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Communicated by M. Takanami
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Love, D.R., Fisher, R. & Bergquist, P.L. Sequence structure and expression of a cloned β-glucosidase gene from an extreme thermophile. Molec. Gen. Genet. 213, 84–92 (1988). https://doi.org/10.1007/BF00333402
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DOI: https://doi.org/10.1007/BF00333402