Salt-inducible promoter derivable from a lactic acid bacterium,

Details Salt-inducible promoter derivable from a lactic acid bacterium, Salt-inducible promoter derivable from a lactic acid bacterium,

1998-07-29

2000-10-31

Yucel, Remy

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435243, 4352523, 4353201, 435 692, 536 231, 536 241, 536 245, C12P 2106

Patent

active

061400785

DESCRIPTION:

BRIEF SUMMARY
This application is the national phase of international application PCT/EP97/04755 filed Aug. 20, 1997 which designated the U.S.
The invention relates to a salt-inducible promoter derivable from a lactic acid bacterium.


BACKGROUND OF THE INVENTION AND PRIOR ART

It is known that salt-inducible promoters exist in plants and cyanobacteria; the latter are rather specialised bacteria which can be used for nitrogen-fixation of soil as natural fertilisation and which taxonomically are quite separate from other bacteria.
Although inducible promoter systems are known in Gram-negative bacteria like E. coli and in the Gram-positive bacterium Bacillus subtilis, while recently in WO 95/31563 (Quest International B.V. (A. Nauta c.s.); see ref. 36) an inducible promoter system was described for lactic acid bacteria or their phages, no literature was found relating to salt-inducible promoters active in microorganisms apart from the above mentioned rather specialised cyanobacteria. Although the expression "salt-initiated induction system" was present in said WO 95/31563 (Quest International B.V. (A. Nauta c.s.); ref. 36), no specific salt-initiated induction system was disclosed.
The present invention provides for the first time a salt-inducible promoter for lactic acid bacteria and its use in the production of polypeptides by lactic acid bacteria.


SUMMARY OF THE INVENTION

The invention provides a salt-inducible promoter derivable from a lactic acid bacterium, in particular a salt-inducible promoter the nucleotide sequence of which is present in SEQ. ID. NO: 10 and in FIGS. 6A-H. The advantage of a salt-inducible promoter active in lactic acid bacteria is first of all that salt is a natural food ingredient and therefore can be used as a food-grade inducer in food fermentation processes. For instance, during the salting stage of cheese curd various processes can be started when the higher salt concentration is used to trigger the formation of various proteins or peptides. Such processes include induced production and secretion of compounds which can contribute to the properties of the final cheese, e.g. enzymes and/or peptides that contribute to the formation of flavour compounds, and lysis of the lactic acid bacteria whereby peptidolytic enzymes are liberated by which the cheese ripening process is enhanced. Another process in which a salt-inducible promoter can be used advantageously is a process for the production of any protein or secondary metabolite by a food-grade microorganism, especially a lactic acid bacterium, whereby at the end of the culturing at a high cell density the microorganism is induced by a salt to produce the desired protein or secondary metabolite. As will be shown in the Examples, in this specification the term "salt" does only not mean common salt, i.e. sodium chloride, but also includes other halides like alkali metal, earth alkali metal and ammonium halides. In addition to the salt-induction by NaCl, KCl, NH.sub.4 Cl, CaCl.sub.2, MgCl.sub.2, NaI, and KI exemplified in Examples 1 and 2 (see also Table 3 below), it is envisaged that also other halides, i.e. bromides and perhaps fluorides, as well as halides with other cations such as substituted ammonium compounds, e.g. tetramethylammonium, or other metallic cations, e.g. Al.sup.3+, will show the salt-inducing effect.
Still another process which can benefit from the presence of a salt-inducible promoter in a microorganism is the production of secondary metabolites, e.g. flavour or taste ingredients, in situ in a fermented product upon the addition of salt, examples of which include dressings and water-containing spreads, as well as sausages and sour dough. The invention also provides a recombinant vector and a transformed lactic acid bacterium each comprising such a salt-inducible promoter.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.
Schematic representation of plasmid pORI13. ori.sup.+, plus origin of pWV01; Em.sup.r, erythromycin resistance gene; T, terminator; lacz, promoterless E. coli .beta.-galactosidase gene fused to the ribosome bind

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