Abstract
Many strains ofBacillus brevis were isolated from nature as very efficient producers of extracellular proteins. Strains identified asB. brevis including these protein-hyperproducers were reclassified into at least 6 species according to numerical analysis, DNA base composition, and DNA-DNA hybridization.
We developed a host-vector system using appropriate strains of theseBacillus brevis as a host, which is excellent for the secretion of heterologous proteins. Utilizing the powerful promoters and signal peptide-coding regions of the cell wall protein gene, various expression-secretion vectors were constructed. The cell wall protein genes of theseB. brevis are transcribed from multiple and tandemly arranged promoters. Transcription from P2, one of the major promoters among them, was enhanced at the early stationary phase of growth, when divalent cations in the medium was depleted and the cell wall protein layers started to be shed. Translation of the cell wall protein gene transcripts starts at the two sites located tandemly in the same reading frame. The two forms of secretory precursors, translation products from the two sites, are cleaved at the same position giving rise to the same mature proteins. The nucleotide sequence from the promoter to the translation start site is highly conserved in protein-hyperproducingB. brevis.
For the efficient secretion of some heterologous proteins, protein-hypersecreting mutants had to be selected. The engineering of the signal peptide was also often necessary to obtain a good secretion of heterologous proteins. Thus, high levels of secretion were achieved only after extensive improvements were made for host, vector and culture conditions. From these experimental results, a great deal of diversity has been observed in various aspects of protein secretion.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Adachi T, Yamagata H, Tsukagoshi N & Udaka S (1989) Multiple and tandemly arranged promoters of the cell wall protein gene operon inBacillus brevis 47. J. Bacteriol. 171: 1010–1016
Adachi T, Yamagata H, Tsukagoshi N & Udaka S (1990) Use of both translation initiation sites of the middle wall protein gene inBacillus brevis 47. J. Bacteriol. 172: 511–513
Adachi T, Yamagata H, Tsukagoshi N & Udaka S (1991) Repression of the cell wall protein gene operon inBacillus brevis 47 by magnesium and calcium ions. J. Bacteriol. 173: 4243–4245
Ebisu S, Tsuboi A, Takagi H, Naruse Y, Yamagata H, Tsukagoshi N & Udaka S (1990) Conserved structures of cell wall protein genes among protein-producingBacillus brevis strains. J. Bacteriol. 172: 1312–1320
Gruber K, Tanahasi H, Tsuboi A, Tsukagoshi N & Udaka S (1988) Comparative study on the cell wall structure of protein-producingBacillus brevis. FEMS Microbiol. letters 56: 113–118
Konishi H, Sato T, Yamagata H & Udaka S (1990) Efficient production of human α-amylase by aBacillus brevis mutant. Appl. Microbiol. Biotechnol. 34: 297–302
Krätzschmar J, Krause M & Marahiel MA (1989) Gramicidin S biosynthesis operon containing the structural genesgrsA andgrsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J. Bacteriol. 171: 5422–5429
Le Grice SF & Sonenshein AL (1982) Interaction ofBacillus subtilis RNA polymerase with a chromosomal promoter. J. Mol. Biol. 162: 551–564
Marahiel MA, Zuber P, Czekay G & Losick R (1987) Identification of the promoter for a peptide antibiotic biosynthesis gene fromBacillus brevis and its regulation inBacillus subtilis. J. Bacteriol. 169: 2215–2222
Mizukami M, Yamagata H, Sakaguchi K & Udaka S (1992) Efficient production of thermostableClostridium thermosulfurogenes β-amylase byBacillus brevis. J. Ferment. Bioengin. 73: 112–115
Shiga Y, Hasegawa K, Tsuboi A, Yamagata H & Udaka S (1992) Characterization of an extracellular protease inhibitor ofBacillus brevis HPD31 and nucleotide sequence of the corresponding gene. Appl. Environ. Microbiol. 58: 525–531
Takagi H, Kadowaki K, Udaka S (1989) Screening and characterization of protein-hyperproducing bacteria without detectable exoprotease activity. Agric. Biol. Chem. 53: 691–699
Takagi H, Shida O, Kadowaki K, Komagata K & Udaka S (1993) Characterization ofBacillus brevis with description ofBacillus migulanus sp. nov.,Bacillus choshinensis sp. nov.,Bacillus parabrevis sp. nov., andBacillus galactophilus sp. nov. Int. J. Syst. Bacteriol. 43: 221–231
Tsuboi A, Tsukagoshi N & Udaka S (1982) Reassemblyin vitro of hexagonal surface arrays in a protein-producing bacterium,Bacillus brevis 47. J. Bacteriol. 151: 1485–1497
Tsuboi A, Uchihi R, Adachi T, Sasaki T, Hayakawa S, Yamagata H, Tsukagoshi N & Udaka S (1988) Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producingBacillus brevis 47: complete nucleotide sequence of the middle wall protein gene. J. Bacteriol. 170: 935–945
Udaka S (1976) Screening for protein-producing bacteria. Agric. Biol. Chem. 40: 523–528
Udaka S & Yamagata H (1993) High-level secretion of heterologous proteins byBacillus brevis. Methods Enzymol. 217: 23–33
Yamada H, Tsukagoshi N & Udaka S (1981) Morphological alterations of cell wall concomitant with protein release in a proteinproducing bacterium,Bacillus brevis 47. J. Bacteriol. 148: 322–332
Yamagata H, Adachi T, Tsuboi A, Takao M, Sasaki T, Tsukagoshi N & Udaka S (1987) Cloning and characterization of the 5' region of the cell wall protein gene operon inBacillus brevis 47. J. Bacteriol. 169: 1239–1245
Yamagata H, Nakagawa K, Tsukagoshi N & Udaka S (1985) A stable plasmid vector and control of its copy number inBacillus brevis 47, a protein-producingbacterium. Appl. Environ. Microbiol. 49: 1076–1079
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Udaka, S., Yamagata, H. Protein secretion inBacillus brevis . Antonie van Leeuwenhoek 64, 137–143 (1993). https://doi.org/10.1007/BF00873023
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00873023