Genetics of Lactic Acid Bacteria:Genetics of proteolysis in Lactococcus lactis

Genetic and biochemical research over the past 15 years on milk protein degradation by Lactococcus lactis has resulted in a detailed picture of how casein is broken down into its sub-fragments and used for cellular growth. Starting with the action of an extracellular but cell-wall-located proteinase, milk protein is degraded into oligopeptides that are internalized by a specialized and essential oligopeptide permease (Opp) system. Without Opp, cells of Lc. lactis cannot grow in milk. Notwithstanding this, the organism has two uptake systems for the internalization of di- and tripeptides. Once inside, a host of intracellular peptidases degrade the oligopeptides into smaller peptides and amino acids that are needed for cell growth. Both general aminopeptidases and more specialized enzymes are involved in this degradation process, as are a number of oligopeptidases. Most of the genes of the components making up the casein degradation pathway have been cloned and sequenced and most of these have been targeted by insertional mutagenesis to allow for an analysis of the function and importance of the components in the pathway. The casein degradation route has been engineered in various ways by the overexpression or stabilization of certain genes, by deleting genes, by mutating enzyme specificities or by expressing heterologous proteolytic enzymes in Lc. lactis. In a number of cases, the modified strains have been used in (small-scale) cheese trials to assess the effects of the changes on the organoleptic quality of the fermentation product. More recently the regulation of the various genes in response to the growth phase of the cells and to medium composition has been studied. Even more recent is the discovery, through the determination of the nucleotide sequence of the chromosome of a widely studied laboratory strain of Lc. lactis, of a number of additional proteinase genes not yet described in this organism. It is no exaggeration to claim that the caseinolytic pathway of Lc. lactis is the best studied of the protein degradation pathways of micro-organisms. This chapter deals with the unravelling of the intricacies of this industrially important catabolic route