Characterization of host factors affecting heterologous protein secretion in Lactococcus lactis.

Lactococcus lactis is the model organism for Lactic Acid Bacteria (LAB) and is a GRAS bacterium. It is therefore a good candidate for the secretion of heterologous proteins of therapeutic interest. In order to investigate new potential uses for LAB, we study host factors involved in the production, the stability and/or the secretion of heterologous proteins in L. lactis. O ur strategy uses a random insertional mutagenesis performed on a L. lactis MG1363 derivative strain carrying a uspnuc cassette in its chromosome. The uspnuc cassette encodes a hybrid precursor composed of the signal peptide o f the Usp45 secreted protein and the staphylococcal nuclease (Nuc) which is used here as a reporter protein for the secretion process. Nuc activity of bacterial colonies is readily detectable in vivo and this provides an effi cient screening for the selection of secretion mutants. We thus selected an d analyzed six L. lactis mutants affected in Nuc secretion. Five out of the 6 corresponding genes were identified. Three of them seem to be involved i n the production and/or secretion of proteins: 2 genes are homologous to un known peptidases and one is homologous to a gene located downstream of a tr anscription regulator. Combination of different strategies should provide n ew insight of the secretion process in L. lactis and could result in the co nstruction of secretion mutants with enhanced capacities.