The use of Pyr-mutants to modify pyrimidine metabolism in lactococci

The specific engineering of organisms used for startercultures has become an effective way of improving and developing new products in the dairy industry. In order to obtain strains with specific characteristics, it is imperative to have a good understanding of the central biochemical pathways . Any growing organism needs nucleotides in order to be able to synthesise DNA, RNA and several co-enzymes.

This demand can be met in two ways 1. By de novo synthesis of nucleotides, or 2. By exploiting nucleotides, nucleosides and nucleobases arising from degradation of DNA or RNA or taken up from the surroundings, the so called salvage pathways. In contrast to the salvage pathways, which may vary between different organisms, the de novo synthesis of pyrimidines seems to be universal.

The pathway consists of six enzymatic reactions leading to UMP, which is subsequently converted into UTP and CTP. If pyrimidines are only available through salvage of uracil and/or cytidine, the pools of CTP and UTP can be varied independently by controlling the activities of cytidine-deaminase (cdd) and CTP-synthetase (pyrG).

Cytidine-deaminase catalyses the deamination of cytidine leading to uridine and CTP-synthetase catalyses synthesis of CTP from UTP. We have cloned the pyrG gene of Lactococcus lactis subsp. cremoris MG1363 and constructed a pyrG mutant that requires cytidine for growth. Furthermore a mutant carrying a chromosomal deletion of the de novo gene pyrF, encoding orotidylat carboxylase, has been constructed.

By combining the two described mutations, with a cdd mutation, we will be able to construct strains in which pyrimidine pools can be manipulated by adding different pyrimidine-sources at variable concentrations to the growth media. Since nucleotides are central metabolites, many physiological parameters may be influenced by the size of the different pyrimidine pools.