Construction of thyA deficient Lactococcus lactis using the Cre-loxP recombination system

Lactococcus lactis is widely used in the food industry as a food-grade non-pathogenic bacterium. In this study, we constructed the thymidylate synthase gene (thyA) deficient strain (NZ9000-3) derived from L. lactis NZ9000 using the Cre-loxP recombination system. Two loxP (lox66 and lox71) sites were integrated into the chromosome at target sites, and the thyA gene was replaced by a chloramphenicol resistance gene (cat) cassette. The recombination between the two loxP sites was very efficient when the Cre recombinase was expressed by a constructed pNZTS-Cre plasmid, and the thyA-null strain was isolated after removal of the cre gene. Then the relationship between the thyA-null strain and a thymidine requirement was analyzed. Results showed that the thyA gene was successfully knocked out, and the thyA-null strain could not grow well in minimal medium. Afterwards, pMG36e-thyA plasmid was constructed and transformed into NZ9000-3. The good screening efficiency revealed that as the selection marker thyA gene was as efficient as the erythromycin resistance gene, this suggested the thyA gene could be used as a food-grade selection marker for L. lactis to be used in food and industry applications.