1887

Abstract

A series of chimeric α-amylase genes derived from , which encodes the liquefying α-amylase from , were constructed using gene splicing techniques. The gene constructs were cloned in , where their ability to direct the synthesis and secretion of active α-amylase was determined. Detectable α-amylase activity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wild-type AmyL and its chimeric derivatives revealed that, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion. The chimeric enzymes were degraded to a greater extent than the native enzyme. These findings suggest that cell-associated proteolysis is a significant problem affecting the use of as host bacterium for the production of heterologous proteins.

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2000-10-01
2020-04-02
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References

  1. Boguslawski G., Grosch J., Schultz J. W., Subramanian S.. 1996; Proteases of altered stability to autolytic degradation. US Patent 5543302
  2. Bolhuis A., Tjalsma H., Smith H. E., de Jong A., Meima R., Venema G., Bron S., van Dijl J.-M.. 1999a; Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis. Appl Environ Microbiol65:2934–2941
    [Google Scholar]
  3. Bolhuis A., Tjalsma H., Stephenson K., Harwood C. R., Venema G., Bron S., van Dijl J.-M.. 1999b; Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase. J Biol Chem274:15865–15868[CrossRef]
    [Google Scholar]
  4. Bron S.. 1991; Plasmids. In Molecular Biological Methods for Bacillus pp.146–147Edited by Harwood C. R., Cutting S. M.. Chichester: Wiley;
    [Google Scholar]
  5. Bron S., Luxen E.. 1985; Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis. Plasmid14:235–244[CrossRef]
    [Google Scholar]
  6. Chambert R., Haddaoui E. A., Petit-Glatron M.-F.. 1995; Bacillus subtilis levansucrase: the efficiency of the second stage of secretion is modulated by external effectors assisting folding. Microbiology141:997–1005[CrossRef]
    [Google Scholar]
  7. Diderichsen B., Outtrup H., Schuein M., Norman B. E.. 1987; Chimeric enzymes. European Patent Application EP-87305781
  8. Diderichsen B., Wedsted U., Hedegaard L., Jensen B. R., Sjøholm C.. 1990; Cloning of aldB, which encodes α-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J Bacteriol172:4315–4321
    [Google Scholar]
  9. Ferrari E., Jarnagin A. S., Schmidt B. F.. 1993; Commercial production of extracellular enzymes. In Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics pp.917–937Edited by Sonenshein A. L., Hoch J. A., Losick R.. Washington: American Society for Microbiology;
    [Google Scholar]
  10. Gray G. L., Mainzer S. E., Rey M. W., Lamsa M. H., Kindle K. L., Carmona C., Requadt C.. 1986; Structural genes encoding the thermophilic α-amylase of Bacillus stearothermophilus and Bacillus licheniformis. J Bacteriol166:635–643
    [Google Scholar]
  11. Harwood C. R.. 1992; Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol10:247–256[CrossRef]
    [Google Scholar]
  12. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R.. 1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene77:61–68[CrossRef]
    [Google Scholar]
  13. Jacobs M., Andersen J. B., Kontinen V., Sarvas S.. 1993; Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesised with or without pro-sequence. Mol Microbiol8:957–966[CrossRef]
    [Google Scholar]
  14. Jensen C. L.. 1997; Secretion of chimeric α-amylases from Bacillus subtilis PhD thesis Technical University of Denmark (DTU);
    [Google Scholar]
  15. Jørgensen P. L., Hansen C. K., Poulsen G. B., Diderichsen B.. 1990; In vivo genetic engineering: homologous recombination as a tool for plasmid construction. Gene96:37–41[CrossRef]
    [Google Scholar]
  16. Jørgensen P. L., Poulsen G. B., Diderichsen B.. 1991a; Cloning of a chromosomal α-amylase gene from Bacillus stearothermophilus. FEMS Lett77:271–276
    [Google Scholar]
  17. Jørgensen S., Skov K. W., Diderichsen B.. 1991b; Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomonasgenes. J Bacteriol173:559–567
    [Google Scholar]
  18. Kieser T.. 1984; Factors affecting the isolation of ccc DNA from Streptomyces lividans and Escherichia coli. Plasmid12:19–36[CrossRef]
    [Google Scholar]
  19. Kontinen V. P., Sarvas M.. 1993; The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high-level secretion. Mol Microbiol8:727–737[CrossRef]
    [Google Scholar]
  20. Kontinen V. P., Saris P., Sarvas M.. 1991; A gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export. Mol Microbiol5:1273–1283[CrossRef]
    [Google Scholar]
  21. Laoide B. M., Chambliss G. H., McConnell D. J.. 1989; Bacillus licheniformis α-amylase gene, amyL, is subject to promoter-independent catabolite repression in Bacillus subtilis. J Bacteriol171:2435–2442
    [Google Scholar]
  22. Leloup L., Haddaoui E. A., Chambert R., Petit-Glatron M.-F.. 1997; Characterization of the rate-limiting step of the secretion of Bacillus subtilis α-amylase overproduced during the exponential phase of growth. Microbiology143:3295–3303[CrossRef]
    [Google Scholar]
  23. Machius M., Wiegand G., Huber R.. 1995; Crystal structure of calcium-depleted Bacillus licheniformis α-amylase at 2·2 Å resolution. J Mol Biol246:545–559[CrossRef]
    [Google Scholar]
  24. Maniatis T., Fritsch E. F., Sambrook J.. 1982; Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  25. Meens J., Herbort M., Klein M., Freudl R.. 1997; Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different Gram-positive bacteria. Appl Environ Microbiol63:2814–2820
    [Google Scholar]
  26. Ortlepp S. A., Ollington J. F., McConnell D. J.. 1983; Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable α-amylase. Gene23:267–276[CrossRef]
    [Google Scholar]
  27. Palva I.. 1982; Molecular cloning of an α-amylase gene from Bacillus amyloliquefaciens and its expression in Bacillus subtilis. Gene10:81–87
    [Google Scholar]
  28. Petit-Glatron M.-F., Graycar L., Munz A., Chambert R.. 1993; The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion. Mol Microbiol9:1097–1106[CrossRef]
    [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R.. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A74:5463–5467[CrossRef]
    [Google Scholar]
  31. Simonen M., Palva I.. 1993; Protein secretion in Bacillus species. Microbiol Rev57:109–137
    [Google Scholar]
  32. Sloma A., Rufo G. A., Rudolph C. F., Sullivan B. J., Theriault K. A., Pero J.. 1989; Cloning and deletion of the genes for three minor extracellular proteases of Bacillus subtilis. In Genetics and Biotechnology of Bacilli pp.295–302Edited by Zukowski M. M., Ganesan A. T., Hoch J. A.. London & New York: Academic Press;
    [Google Scholar]
  33. Sloma A., Rufo G. A., Theriault K. A., Dwyer M., Wilson S. W., Pero J.. 1991; Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis. J Bacteriol173:6889–6895
    [Google Scholar]
  34. Spizizen J.. 1958; Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci U S A44:1072–1078[CrossRef]
    [Google Scholar]
  35. Stephenson K., Harwood C. R.. 1998; The influence of a cell-wall-associated protease on the production of α-amylase by Bacillus subtilis. Appl Environ Microbiol64:2875–2881
    [Google Scholar]
  36. Stephenson K., Carter N. M., Harwood C. R., Petit-Glatron M.-F., Chambert R.. 1998; The influence of protein folding on the secretion of α-amylase from Bacillus subtilis. FEBS Lett430:385–389[CrossRef]
    [Google Scholar]
  37. Stephenson K., Bron S., Harwood C. R.. 1999; Cellular lysis in Bacillus subtilis: the effect of multiple extracellular protease deficiencies. Lett Appl Microbiol29:141–145[CrossRef]
    [Google Scholar]
  38. Stephenson K., Jensen C., Jørgensen S. T., Lakey J. H., Harwood C. R.. 2000; Characterisation and properties of chimeric α-amylases produced in Bacillus subtilis. Biochem J in press
    [Google Scholar]
  39. Takkinen K., Pettersson R. F., Kalkkinen N., Palva I., Söderlund H., Kääriäinen L.. 1983; Amino acid sequence of α-amylase from Bacillus amyloliquefaciens deduced from the nucleotide sequence of the cloned gene. J Biol Chem258:1007–1013
    [Google Scholar]
  40. Wu X.-C., Lee W., Tran L., Wong S.-L.. 1991; Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. J Bacteriol173:4952–4958
    [Google Scholar]
  41. Yanisch-Perron C., Vieira J., Messing J.. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene33:103–119[CrossRef]
    [Google Scholar]
  42. Yasbin R. E., Wilson G. A., Young F. E.. 1975; Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J Bacteriol121:296–304
    [Google Scholar]
  43. Yoneda Y., Graham S., Young F. E.. 1979; Cloning of foreign gene for α-amylase in Bacillus subtilis AHU1031. Eur J Biochem141:83–89
    [Google Scholar]
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