Abstract: The invention provides a fed-batch 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: A method of identifying nucleotide sequences coding for signal peptides in lactic acid bacteria, using a DNA molecule comprising a transposon including a promoterless reporter gene from which DNA molecule a region between the LR and the reporter gene is deleted and the DNA molecule comprises a DNA sequence coding for a secretion reporter molecule. By deleting the region between the LR and the reporter gene, stop codons in-frame with the secretion reporter molecule is removed which upon transposition permits translational fusions from upstream the LR.

Abstract: A method of identifying nucleotide sequences coding for signal peptides in lactic acid bacteria, using a DNA molecule comprising a transposon including a promoterless reporter gene from which DNA molecule a region between the LR and the reporter gene is deleted and the DNA molecule comprises a DNA sequence coding for a secretion reporter molecule. By deleting the region between the LR and the reporter gene, stop codons in-frame with the secretion reporter molecule is removed which upon transposition permits translational fusions from upstream the LR.

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.