Peptides of interest: Editing of Lactococcus lactis proteolytic system to increase its bioactive potential

The goal of this thesis was to answer one question in particular: can one increase the quantity or the diversity (or both) of milk-derived bioactive peptides by engineering the Lactococcus lactis proteolytic system? The main research line explored that question itself. In addition, the three issues that derive from that main questions are separately tackled in this thesis: (i) bioactive peptides: How much do we know about bioactive peptides derived from milk-derived and, more specifically, beta-casein? (ii) L. lactis engineering: Which tools are there and are they good enough or can we develop new/better ones; (iii) 3. The L. lactis proteolytic system: what do we know about that system and, especially, what do we know about the in vivo (complementing) activities of the peptidases with respect to cellular growth and peptide degradation Chapter 1 comprehensively reviews the knowhow on β-casein-derived bioactive peptides and the potential of using lactic acid bacteria to produce such peptides; In Chapter 2, which is the founding chapter of the thesis, we engineered the L. lactis proteolytic system by making a large collection of various combinations of peptidase gene mutants and used those mutants to increase the quantity of different bioactive peptides and the diversity of different bioactivities which derived from β-casein; In Chapter 3, we broaden the knowledge about the L. lactis proteolytic system, and prove that the dipeptidase PepV plays an important role in peptidoglycan biosynthesis by acting as a link between nitrogen metabolism and cell wall synthesis. In Chapter 4, we expand the genetic toolbox for L. lactis by developing plasmid- and genome-based CRISPRi systems that will allow rapidly e.g., editing biological pathways or characterizing essential genes, as was explored in Chapter 5