Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-07-15
2004-09-14
Slobodyansky, Elizabeth (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S252300, C435S320100, C530S324000, C530S326000
Reexamination Certificate
active
06790951
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to bacteriocins that are produced and secreted by various lactic acid bacteria. More specifically, it relates to the use of regulatory genetic elements (promoters) belonging to the genes that are involved in the production and secretion of bacteriocins. More specifically, it relates to the use of these genetic elements for the regulated expression of homologous or heterologous genes in lactic acid bacteria, in particular members of the genus Lactobacillus. Genes that have been manipulated in such a way as to put their expression under control of the invented genetic elements, as well as cells and vectors comprising such manipulated genes and a kit are also part of the present invention.
Cells produce proteins according to genetic information that is contained in the DNA. Many of these proteins catalyze chemical reactions in or outside the cells and are called enzymes. A piece of DNA that contains all the information for a protein is called a gene. A gene (DNA) is expressed as follows: first the process of transcription results in the formation of a so-called messenger RNA (mRNA); subsequently, in the process of translation, a protein (sometimes simply referred to as the gene-product) is synthesized, using the information contained in the mRNA.
To enable transcription, a gene is preceded by a promoter (=a piece of DNA preceding the gene that is not directly encoding the geneproduct but that is essential for initiation of transcritpion). In the process of transcription, the key enzyme involved in this process, RNA polymerase, associates with the promoter and, subsequently, a messenger RNA is synthesized. The extent to which a gene is expressed is often regulated at the transcriptional level; in other words, the level of transcription is regulated and this regulates the level at which the gene product is produced. Genes which are transcribed at a fixed (non-regulated) rate are said to be expressed constitutively. Regulation of transcription can be effectuated by regulating the association between the promoter and the RNA-polymerase. To achieve the latter a promoter element may contain stretches of DNA that 1) facilitate or impair association of RNA polymerase, or 2) permit the binding of additional factors (transcription factors) that enhance (‘activator’) or inhibit (‘repressor’) the binding of the RNA polymerase. In an alternative way of regulating gene transcription, the effectiveness of an associated RNA polymerase is regulated by binding of additional factors that, for example, prevent the associated RNA polymerase from actually starting the synthesis of an RNA molecule.
Bacteria can be made to produce certain proteins by introducing (‘cloning’) the corresponding gene in one or more copies. The gene may be maintained in the host organism either by using a plasmid, the vector, or by integrating it in the chromosome. To really obtain production of the desired geneproduct the cloned gene needs to be expressed. In most cases, this means that additional genetic engineering is necessary to put the cloned gene under the control of a specific promoter element that is recognized by the host organism. Such promoters may be regulable. These techniques of genetic manipulation can be used to establish two types of ‘engineered’ expression:
Homologous expression: an organism expresses a gene from the same species. The level of expression may for example be regulated by regulating the number of copies of the gene present per cell or by varying the promoter element by which the transcription of the gene is controlled.
Heterologous expression: as above, but now the gene that is expressed comes from a different species.
The establishment of the (regulable) expression of homologous or heterologous genes is an important part of genetic engineering in the biotechnological industry.
Temporal and quantitative regulation of gene expression is an important aspect of genetic engineering as employed in the biotechnological industry. Regulation can be an important problem, both in terms of timing of the expression and in terms of the quantity of expression. Really well regulable promoters applicable in industrially important micro-organisms are therefore of major interest.
Lactic acid bacteria (LAB), such as members of the bacterial genera Lactococcus, Lactobacillus and Pediococcus are of major importance in fermentations conducted in the food and feed industry. Many LAB have the GRAS (Generally Regarded As Safe) status and they are consumed by humans regularity and in large amounts (for example in dairy products).
Many LAB produce bacteriocins, anti-microbial peptides that are harmless to humans (1,2). These peptides normally contain between 30 and 60 residues, they usually have a basic character, and, often, parts of their sequence show amphiphilicity when projected onto a helical wheel. Some bacteriocins undergo post-translational modification and are called lantibiotics. Bacteriocins are produced as precursor proteins with a leader peptide that is removed during export.
An expression system for Lactobacillus, Lactococcus and Bacillus is previously described in WO no. 94/00581 (VIAGEN OY). This expression system employs expression signals (promoters) linked to coat protein expression in Lactobacillus in combination with various secretion signals. This expression system does not employ regulable promoters. It provides some means for quantitative regulation of gene expression that are different from the means described in the present invention. It does not provide means for temporal regulation of gene expression. It is not based on promoters and regulatory mechanisms linked to bacteriocin production.
It is further published a paper by Djodevic, G. et al., “Cloning of promoter like sequences . . .
Lactobacillus paracasei
subsp. . . . ”, Can. J. Microbiol. (1994), 40 (12), 1043-50. This publication relates to gene expression in Lactobacillus but does not describe or suggest means to regulate gene expression temporarily, as is done in the present invention. Furthermore, this publication concerns normal, non-regulable promoters that give raise to constitutive, non-regulable expression in Lactobacillus.
It is further published a paper by Tizacheck, P. S., Vogel, R. F. and Hammes, W. P., “Cloning and sequencing of SAKP encoding sakacin-P, the bacteriocin produced by
Lactobacillus sake
LTH673”, Microbiology-UK (1994) V140, FEB (FEB), 361-67, and there is an entry in the EMBL/GenBank/DDBJ database (accession number Z48542)(SEQ ID NOS: 13-19) by Huehne, K., Holck, A., Axelsson L. and Kroeckel, L. (1995). These two publications describe the nucleotide sequences of pieces of DNA from, respectively,
Lactobacillus sake
LTH673, and
Lactobacillus sake
Lb674, that are, within the experimental error, identical and that encode genes involved in the production of bacteriocin, called sakacin P. These nucleotide sequences do also contain the promoter sequences depicted in
FIG. 4
(the upper four sequences). However, these publications do only describe sequences; they do not describe: 1) the regulatory mechanism for gene expression that is part of the present invention, 2) the promoter elements involved in this regulatory mechanism that are part of the present invention, 3) the expression-inducing peptides that are part of the present invention, 4) possible applications of the regulatory mechanisms referred to in points 1-3. In other words these publications do not relate to the regulation of gene expression in Lactobacillus by use of specific regulable promoters, nor do they describe such promoters. In other words: knowing the sequences of genes involved in the production of bacteriocins, including the sequences upstream of those genes (that contain promoter elements) is in itself not enough to recognize (1) the regulatory mechanism involved in the regulation of the expression of genes involved in bacteriocin production, (2) the regulable promoter elements involved, or (3) the biotechnological potential of this regulatory mechanism.
In N. Balaban an
Brurberg May B.
Eijsink Vincent G. H.
Nes Ingolf F.
Birch & Stewart Kolasch & Birch, LLP
Slobodyansky Elizabeth
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