Complex inducible promoter system derivable from a phage of a la

Details Complex inducible promoter system derivable from a phage of a la Complex inducible promoter system derivable from a phage of a la

1997-04-03

2000-11-07

Yucel, Remy

Chemistry: molecular biology and microbiology

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

Using a micro-organism to make a protein or polypeptide

435 691, 435 711, 4352523, 4353201, 536 241, C12P 2104

Patent

active

061435252

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to a complex inducible promoter system derivable from a phage of a lactic acid bacterium.
2. Background of the Invention and Prior Art
Although complex inducible promoter systems are known in Gram-negative bacteria like E. coli and in the Gram-positive bacterium Bacillus subtilis, no such promoter system has been described for lactic acid bacteria or their phages.
The invention is directed to an inducible strong promoter for lactic acid bacteria, which preferably can be induced in a food-grade manner. Although various constitutive, weak and strong, promoters for lactic acid bacteria are known, there is still a need for an inducible promoter effective in lactic acid bacteria.


BRIEF SUMMARY OF THE INVENTION

The invention provides a complex inducible promoter system derivable-from a phage of a lactic acid bacterium, in particular a complex inducible promoter system comprising the DNA sequence given in FIG. 2 and SEQ. ID. NOS: 3-6 or a functionally equivalent DNA sequence. A suitable embodiment is formed by a DNA sequence essentially corresponding to the DNA sequence of sequence id no 3. Other alternative embodiments comprise at least an essential part of such a complex inducible promoter system such as the DNA sequence given in FIG. 3 (which is the part of FIG. 2 or sequence id. no. 3 lacking ORF 29 and its ribosome binding site RBS). The promoter system can be modified by replacing the mitomycin C induction system by a food-grade system, such as a temperature-initiated induction system or a salt-initiated induction system by mutation techniques known per se.
"A functional equivalent promoter system to that of sequence id no 3" is understood as to include variants and mutants that have a different nucleic acid sequence but form a functional inducible complex promoter which is capable of regulating expression of a gene operatively linked to it under inducing circumstances or after having undergone induction to a degree comparable to the specifically disclosed sequences forming a complex inducible promoter system according to the invention.
For example the nucleic acid sequences linking and flanking certain essential elements of the complex promoter system required for it to function as an inducible promoter can be different to those in the disclosed nucleic acid sequence id no 3. Preferably the length of such linking and flanking sequences will be the same as in the disclosed sequences in order to maintain a similar structure to that of the disclosed sequences of sequence id no 3. The spacing and orientation of the elements should be preferably be the same as of the nucleic acid sequences in sequence id no. 3. The essential elements are a combination of operator sequences 1 and 2, the repressor gene (ORF 27), the topological equivalent of cro gene (ORF 28) and the ribosomal binding sites of the corresponding genes, the SD sequence and the promoters of the genes.
A number of variations or mutations that can be considered to be obvious to a person skilled in the art in comparison to the illustrated nucleic acid sequences whilst still retaining the functionality of the complex inducible promoter and thus being considered functionally equivalent will now be presented. Any variations or mutations of the complex inducible promoter system encoded by the nucleic acid sequence not impairing the expression capacity of the promoter system or the foodgrade status is considered to fall within the scope of the invention. With regard to the expression products of ORF 27, ORF 28 and ORF 29 it is obvious that various nucleic acid sequences encode the same amino acid sequences of these expression products other than the nucleic acid sequence of sequence id no 3 or segments 880-1654, 390-350, 336-1 of Sequence id no 3. Simply by substituting one or more codons encoding the same amino acid a nucleic acid sequence different to that of Sequence id no 3 can be obtained which will be functionally equivalent to the complex inducible promoter system

REFERENCES:
van der Vossen et al, Applied and Environmental Microbiology, vol. 53, No. 10, pp. 2452-2457 (Oct. 1987).
Platteeuw et al, Applied and Environmental Microbiology, vol. 60, No. 2, pp. 587-593 (Feb. 1994).
Lakshmidevi et al, Applied and Environmental Microbiology, vol. 56, No. 4, pp. 934-942 (Apr. 1990).
van de Guchte et al, Gene, 144 (1994) 93-95.
Achen et al, Gene, 45 (1986) 45-59.
Kim et al, Food Microbiology, 8 (1991) 27-36.
van de Guchte et al, FEMS Microbiology Reviews, 88 (1992) 73-92.

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