Development of a counterselectable seamless mutagenesis system in lactic acid bacteria

Background Lactic acid bacteria (LAB) are receiving more attention to act as cell factories for the production of high-value metabolites. However, the molecular tools for genetic modifying these strains are mainly vector-based double-crossover strategies, which are laborious and inefficient. To address this problem, several counterselectable markers have been developed, while few of them could be used in the wild-type host cells without pretreatment. Results The pheS gene encoding phenylalanyl-tRNA synthetase alpha subunit was identified in Lactococcus lactis NZ9000 genome. When mutant pheS gene ( pheS* ) under the control of the Lc. lactis NZ9000 l -lactate dehydrogenase promoter (P ldh ) was expressed from a plasmid, the resulted PheS* with an A312G substitution rendered cells sensitive to the phenylalanine analog p -chloro-phenylalanine ( p -Cl-Phe). This result suggested pheS* was suitable to be used as a counterselectable marker in Lc. lactis . However, the expression level of pheS* from a chromosomal copy was too low to confer p -Cl-Phe sensitivity. Therefore, a strategy of cascading promoters was attempted for strengthening the expression level of pheS* . Expectedly, a cassette 5Pldh- pheS* with five tandem repetitive promoters P ldh resulted in a sensitivity to 15 mM p -Cl-Phe. Subsequently, a counterselectable seamless mutagenesis system PheS*/pG + host9 based on a temperature-sensitive plasmid pG + host9 harboring a 5Pldh- pheS* cassette was developed in Lc. lactis . We also demonstrated the possibility of applying pheS* to be a counterselectable marker in Lactobacillus casei BL23. Conclusions As reported in E. coli , pheS* as a counterselectable marker has been demonstrated to be functional in targeted gene(s) deletion in Lc. lactis as well as in L. casei . Moreover, the efficiency and timesaving counterselectable seamless mutagenesis system PheS*/pG + host9 could be used in the wild-type host cells without pretreatment.