Abstract: Nucleotide sequences coding for a promoter activity-regulating polypeptide designated OrfY, isolated from Lactococcus lactis, and promoter activity-regulating active homologues and fragments hereof is used to indirectly regulating the expression of genes. The coding sequence is inserted into lactic acid bacterial cells or any other cells with the objective of either reducing or enhancing the expression level of the gene being under the control of the promoter sequence, the activity of which is regulated by the OrfY polypeptide or homologues or fragments hereof. Improved gene expression systems are provided based on this promoter activity-regulating polypeptide.

Abstract: or continuous process for producing heterologous peptides, polypeptides or proteins in lactic acid bacteria, comprising the steps of (i) constructing a recombinant lactic acid bacterium comprising a nucleotide sequence coding for the heterologous peptide, polypeptide or protein, and appropriate regulatory nucleotide sequences such as regulatable promoters and signal peptides to control the expression of the coding sequence and the secretion of gene product, (ii) cultivating the recombinant bacterium under or continuous cultivation conditions to express the gene, and (iii) harvesting the recombinant bacterium or the gene product. Preferably, the cultivation medium is a chemically defined or synthetic medium optionally supplemented with yeast extract.

Abstract: Expression vectors capable of being replicated in lactic acid bacterial cells comprising a promoter region comprising (a) a promoter sequence element the function of which is regulatable by an environmental or growth condition factor and (b) at least one further nucleotide sequence element, the position, orientation, presence and/or sequence of which element has a regulatory effect on the expression of a gene operably linked to the promoter region in which vectors the position, orientation, presence and/or sequence of at least one of said elements (a) or (b) is modified relative to the position, orientation, presence and/or sequence of the corresponding non-modified element whereby the expression of the gene is altered, and a lactic acid bacterium which is transformed with such a vector as defined above are provided.

Abstract: A method of isolating a lactic acid bacterial DNA fragment comprising a promoter, the method comprising introducing a DNA molecule comprising a transposable element comprising a promoterless structural gene as a promoter probe gene into a population of a lactic acid bacterium, methods of constructing a recombinant lactic acid bacterium comprising a regulatable promoter by using the above method, a recombinant lactic acid bacterium comprising a gene coding for a desired gene product and operably linked thereto a regulatable lactic acid bacterial promoter not natively associated with the gene, the use of such a recombinant lactic acid bacterium and recombinant plasmids comprising a regulatable lactic acid bacterial promoter.