Dairy cultures with modified nucleotide metabolism

Lactococcus lactis is a facultativ anaerobic gram positive bacterium, widely used in the dairies, and - at least by mass - the most important organism used in milk fermentations. As all other organisms, they need nucleotides in order to grow. Nucleotides are not only precursors in the synthesis of RNA og DNA, but are constituting vital parts of coenzymes in the biosynthesis of proteins, phospholipides og polysaccharides. Furthermore, nucleotides acts as allosteric effectors in a large number of enzymatic reactions, and due to their role as energy transporters, they are directly involved in the different metabolic pathways, including stress response and glycolysis. In most organisms nucleotides are made either de novo or from prefabricated nucleosides og nucleobases, already present in the growth medium or formed intracellulary from nucleic acid turnover, through the socalled salvage pathways.
The balance of a number of different activities in the living cell, is dependent on the concentration of different nucleotides. By shifting the the pool sizes we expect the cell to respond by altering their physiological state. The aim of this project is to construct strains with altered CTP and UTP pools and analyze the effect on the production of macromolecules and metabolites. The gene encoding CTP synthetase (pyrG) will be cloned, and by creating a pyrG mutation in a strain lacking the cytidine deminase activity, the CTP and UTP pools will be totally separated. In combination with a pyrimidine requiring strain, the internal concentrations of UTP and CTP can now be controlled by feeding with limiting amounts of uracil and/or cytidine. The production of macromolecules and metabolites of the strains growing under these conditions wil be analyzed in order to monitor the physiological state of these cells.