Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Aug;173(16):4952–4958. doi: 10.1128/jb.173.16.4952-4958.1991

Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases.

X C Wu 1, W Lee 1, L Tran 1, S L Wong 1
PMCID: PMC208183  PMID: 1907264

Abstract

We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride. By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.

Full text

PDF
4952

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amory A., Kunst F., Aubert E., Klier A., Rapoport G. Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis. J Bacteriol. 1987 Jan;169(1):324–333. doi: 10.1128/jb.169.1.324-333.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aymerich S., Steinmetz M. Cloning and preliminary characterization of the sacS locus from Bacillus subtilis which controls the regulation of the exoenzyme levansucrase. Mol Gen Genet. 1987 Jun;208(1-2):114–120. doi: 10.1007/BF00330431. [DOI] [PubMed] [Google Scholar]
  3. Chang S. Engineering for protein secretion in gram-positive bacteria. Methods Enzymol. 1987;153:507–516. doi: 10.1016/0076-6879(87)53075-0. [DOI] [PubMed] [Google Scholar]
  4. Crutz A. M., Steinmetz M., Aymerich S., Richter R., Le Coq D. Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system. J Bacteriol. 1990 Feb;172(2):1043–1050. doi: 10.1128/jb.172.2.1043-1050.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Henner D. J., Ferrari E., Perego M., Hoch J. A. Location of the targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in upstream regions of the subtilisin promoter. J Bacteriol. 1988 Jan;170(1):296–300. doi: 10.1128/jb.170.1.296-300.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J Bacteriol. 1982 May;150(2):804–814. doi: 10.1128/jb.150.2.804-814.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kawamura F., Doi R. H. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol. 1984 Oct;160(1):442–444. doi: 10.1128/jb.160.1.442-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Koide Y., Nakamura A., Uozumi T., Beppu T. Cloning and sequencing of the major intracellular serine protease gene of Bacillus subtilis. J Bacteriol. 1986 Jul;167(1):110–116. doi: 10.1128/jb.167.1.110-116.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Le Grice S. F. Regulated promoter for high-level expression of heterologous genes in Bacillus subtilis. Methods Enzymol. 1990;185:201–214. doi: 10.1016/0076-6879(90)85020-o. [DOI] [PubMed] [Google Scholar]
  10. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  11. Ochi K., Kandala J. C., Freese E. Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation. J Biol Chem. 1981 Jul 10;256(13):6866–6875. [PubMed] [Google Scholar]
  12. Osburne M. S., Craig R. J., Rothstein D. M. Thermoinducible transcription system for Bacillus subtilis that utilizes control elements from temperate phage phi 105. J Bacteriol. 1985 Sep;163(3):1101–1108. doi: 10.1128/jb.163.3.1101-1108.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Palva I., Sarvas M., Lehtovaara P., Sibakov M., Käriäinen L. Secretion of Escherichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5582–5586. doi: 10.1073/pnas.79.18.5582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Priest F. G. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev. 1977 Sep;41(3):711–753. doi: 10.1128/br.41.3.711-753.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  16. Shimotsu H., Henner D. J. Modulation of Bacillus subtilis levansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes. J Bacteriol. 1986 Oct;168(1):380–388. doi: 10.1128/jb.168.1.380-388.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sloma A., Ally A., Ally D., Pero J. Gene encoding a minor extracellular protease in Bacillus subtilis. J Bacteriol. 1988 Dec;170(12):5557–5563. doi: 10.1128/jb.170.12.5557-5563.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sloma A., Rudolph C. F., Rufo G. A., Jr, Sullivan B. J., Theriault K. A., Ally D., Pero J. Gene encoding a novel extracellular metalloprotease in Bacillus subtilis. J Bacteriol. 1990 Feb;172(2):1024–1029. doi: 10.1128/jb.172.2.1024-1029.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sloma A., Rufo G. A., Jr, Rudolph C. F., Sullivan B. J., Theriault K. A., Pero J. Bacillopeptidase F of Bacillus subtilis: purification of the protein and cloning of the gene. J Bacteriol. 1990 Mar;172(3):1470–1477. doi: 10.1128/jb.172.3.1470-1477.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. doi: 10.1073/pnas.44.10.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stahl M. L., Ferrari E. Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation. J Bacteriol. 1984 May;158(2):411–418. doi: 10.1128/jb.158.2.411-418.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Steinmetz M., Le Coq D., Aymerich S., Gonzy-Tréboul G., Gay P. The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites. Mol Gen Genet. 1985;200(2):220–228. doi: 10.1007/BF00425427. [DOI] [PubMed] [Google Scholar]
  23. Wang P. Z., Doi R. H. Overlapping promoters transcribed by bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. J Biol Chem. 1984 Jul 10;259(13):8619–8625. [PubMed] [Google Scholar]
  24. Wong S. L. Development of an inducible and enhancible expression and secretion system in Bacillus subtilis. Gene. 1989 Nov 30;83(2):215–223. doi: 10.1016/0378-1119(89)90107-8. [DOI] [PubMed] [Google Scholar]
  25. Wong S. L., Doi R. H. Determination of the signal peptidase cleavage site in the preprosubtilisin of Bacillus subtilis. J Biol Chem. 1986 Aug 5;261(22):10176–10181. [PubMed] [Google Scholar]
  26. Wong S. L., Kawamura F., Doi R. H. Use of the Bacillus subtilis subtilisin signal peptide for efficient secretion of TEM beta-lactamase during growth. J Bacteriol. 1986 Nov;168(2):1005–1009. doi: 10.1128/jb.168.2.1005-1009.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wong S. L., Price C. W., Goldfarb D. S., Doi R. H. The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1184–1188. doi: 10.1073/pnas.81.4.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wu X. C., Nathoo S., Pang A. S., Carne T., Wong S. L. Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from Bacillus subtilis. J Biol Chem. 1990 Apr 25;265(12):6845–6850. [PubMed] [Google Scholar]
  29. Yang M. Y., Ferrari E., Henner D. J. Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation. J Bacteriol. 1984 Oct;160(1):15–21. doi: 10.1128/jb.160.1.15-21.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yansura D. G., Henner D. J. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci U S A. 1984 Jan;81(2):439–443. doi: 10.1073/pnas.81.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zukowski M. M., Miller L., Cosgwell P., Chen K., Aymerich S., Steinmetz M. Nucleotide sequence of the sacS locus of Bacillus subtilis reveals the presence of two regulatory genes. Gene. 1990 May 31;90(1):153–155. doi: 10.1016/0378-1119(90)90453-x. [DOI] [PubMed] [Google Scholar]
  32. Zukowski M. M., Miller L. Hyperproduction of an intracellular heterologous protein in a sacUh mutant of Bacillus subtilis. Gene. 1986;46(2-3):247–255. doi: 10.1016/0378-1119(86)90409-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES