The goal of this proposal is to elucidate the exact configuration and role of the 2-CS CesSR in L. lactis (stress) physiology. The ultimate aim of the work on L. lactis protein production is to develop the organism into a legitimate alternative host for the production of (medically relevant) membrane proteins. These objectives will be pursued through a project with four interconnected work packages (WPs): [WP1] The role of sRNA in membrane protein overproduction stress, will be determined studied the transcriptomes of MPs overproducing L.lactis strains obtained by RNA sequencing. [WP2] Unravel the regulon of cesFSR and the role of cesF in cesR activity. The effects of mutations in cesS, cesR as well as cesF on the response of L. lactis to MP overproduction will be examined through RNA sequencing. [WP3] The establishment of the roles of identified sRNAs in cesFSR-mediated stress response will be determined. For this purpose, a selection of the sRNAs positively identified in WP1 and WP2 will also be subjected to in-depth mutational analysis to uncover their regulatory roles. [WP4] The obtaining of L.lactis strains with improved MP production characteristics. These strains will be constructed according to the results of WP1, WP2 and WP3. Results from this proposal hold promise for further increasing MP overproduction capacity of L. lactis, solving some of the long-standing issues in this field of research, and for creating intellectual property rights for a high-tech small or medium-sized enterprise in the biotechnology/pharmaceutical field.