Chemistry: molecular biology and microbiology – Vector – per se
Patent
1997-06-06
1999-08-17
Degen, Nancy
Chemistry: molecular biology and microbiology
Vector, per se
435 6, 435 691, 4351723, 4352529, 536 231, 536 237, 536 241, C12N 1563, C12N 1511, C07H 2104
Patent
active
059393173
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the use of a Sec-dependent secretion system for secreting proteins normally secreted by a Sec-independent secretion system. It further relates to the lactic acid bacteria containing this Sec-dependent secretion system, in particular the lactococci belonging to the species Lactococcus lactis, to the use of certain strains of these lactococci for transferring said secretion system to strains of industrial interest, particularly in the dairy industry, and to the use of certain strains of Lactococcus lactis for obtaining this secretion system.
The secretion of proteins in bacteria generally follows two pathways. The better known is Sec-dependent transport. According to this pathway, the protein is exported by virtue of the presence, in the N-terminal position, of a signal peptide which is cleaved in the secreted protein. Several proteins involved in this secretion, particularly SecA, SecY and SecE, have been identified in E. coli. The secY gene has been identified in L. lactis: it possesses a high degree of homology with the secY gene of E. coli, indicating that this secretion system is very probably also present in L. lactis (Koivula et al., 1991, FEBS Lett. 228: 114-118).
The second secretion pathway in bacteria, which does not depend on the Sec proteins, involves transmembrane translocators called the A, B, C family of proteins (Pugsley, 1993, Microbiol. Rev. 57: 50-108).
Translocators have been identified in L. lactis; they are involved in the secretion of the bacteriocins LcnA, LcnB and LcnMl and the antibiotics lacticin 481, nisin A and nisin Z (de Vos and Simons, Genetics and Biotechnology of L.A.B., Blackie Academic and Professional, 1994).
The study of these two secretion pathways in L. lactis has been utilized to develop different systems for the secretion of heterologous proteins. The secretion vectors developed are all contained in the review by de Vos and Simons, Genetics and Biotechnology of L.A.B., Blackie Academic and Professional, 1994.
The bacteriocins identified in lactic acid bacteria are virtually all secreted by the Sec-independent system (Dodd and Gasson, Genetics and Biotechnology of L.A.B., Blackie Academic and Professional, 1994). However, in the interest of increasing the secretion of these bacteriocins, it can be important to cause secretion of these bacteriocins by a different secretion system.
Some authors have recently shown that divergicin A, a bacteriocin produced by Carnobacterium divergens, is secreted via a Sec-dependent system (Worobo et al., J. Bacteriol. 177: 3143-3149). However, it has never been shown in lactic acid bacteria that a bacteriocin secreted by a Sec-independent system can be secreted by a Sec-dependent system.
The Applicant has carried out studies in this field and has found, surprisingly, that a Sec-dependent secretion system can be used to secrete proteins normally secreted by a Sec-independent secretion system.
According to the invention, the Sec-dependent secretion system is used in combination with the DNA sequence coding for a mature protein normally secreted by a Sec-independent system, and with an appropriate promoter, an appropriate signal sequence recognized by the Sec-dependent secretion system and an appropriate terminator.
Thus the invention further relates to the DNA constructions for the secretion, by a Sec-dependent system, of proteins normally secreted by a Sec-independent system, said DNA constructions comprising a promoter, a signal sequence recognized by the Sec-dependent secretion system, the DNA sequence coding for a mature protein normally secreted by a Sec-independent system, and a terminator. These DNA constructions can be present on an expression vector such as a plasmid, in the genomic DNA or in any other DNA fragment.
According to the invention, any Sec-dependent secretion system can be used in combination with a gene coding for a protein of interest which is normally secreted by a Sec-independent system.
Any Sec-dependent secretion system is suitable for the purposes of the invention. Particularly
REFERENCES:
Oliver et al. "SecA protein: Autoregulated ATPase Catalysing Preprotein Insertion and Translocation Across the Eschericia coli Inner Membrane" Molecular Microbiology vol. 7(2): 159-165, 1993.
M. van de Guchte, "Gene Expression in Lactococcus lactis", FEMS Microbiology Reviews, vol. 88, 1992, pp. 73-92.
J. Kok, et al., "Nucleotide Sequence of the Cell Wall Proteinase Gene of Streptococcus cremoris Wg2", Applied and Environmental Microbiology, vol. 54, No. 1, Jan. 1988, pp. 231-238.
M. van Belkum, et al., "Cloning, Sequencing, and Expression in Escherichia coli of IcnB, a Third Bacteriocin Determinant from the Lactococcal Bacteriocin Plasmid p9B46", Applied and Environmental Microbiology, vol. 58, No. 2, Feb. 1992, pp. 572-577.
O.P. Kuipers, et al., "Expression of Wild-Type and Mutant Nisin Genes in Lactococcus lactis", Editor: G. Jung, et al., "Nisin and Novel Lantibiotics", Proceedings of the First International Workshop on Lantibiotics, Apr. 15-18, 1991, pp. 250-258.
Bigret Marc
Fayard Blandine
Kok Jan
Prevots Fabien
Venema Gerhardus
Degen Nancy
McGarry Sean
SKW Biosystems
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