Construction of Lactococcus lactis thyA -null using the Red recombination system

Lactococcus lactis—a food-grade nonpathogenic lactic acid bacterium—is used widely in the food industry. In this report, we describe an approach to construct deficient strains in L. lactis utilizing the λ-Red recombination system. Three kinds of recombinant proteins, λ exonuclease, β protein and γ protein, were induced by l-arabinose in L. lactis MG1363 harboring the plasmid pKD46. A chloramphenicol-resistant cassette was amplified from pGj103 containing homology arms of 50 nt to the thyA gene. The PCR-generated DNA fragment was then electroporated into L. lactis MG1363, which expressed the recombination proteins. ThyA-null strains resistant to chloramphenicol were obtained and their growth characteristics were analyzed in relation to thymidine requirement. The results revealed that the thyA gene in L. lactis MG1363 was successfully knocked out. This is the first time that the Red system has been used in a Gram-positive bacterium, and use of the techniques presented here should prompt rapid and efficient mutagenesis or modification of L. Lactis chromosomal genes.