title: Generation and characterization of thymidine/D-alanine auxotrophic recombinant Lactococcus lactis subsp. lactis IL1403 expressing BmpB.
abstract: Genetic engineering of Lactococcus lactis to produce a heterologous protein may cause potential risks to the environment despite the industrial usefulness of engineered strains. To reduce the risks, we generated three auxotrophic recombinant L. lactis subsp. lactis IL1403 strains expressing a heterologous protein, BmpB, using thyA- and alr-targeting integration vectors: ITD (thyA(-)alr(+) bmpB(+)), IAD (thyA(+)alr(-)bmpB(+)), and ITDAD (thyA(-)alr(-) bmpB(+)). After construction of integration vectors, each vector was introduced into IL1403 genome. Integration of BmpB expression cassette, deletion of thyA, and inactivation of alr were verified by using PCR reaction. All heterologous DNA fragments except bmpB were eliminated from those recombinants during double crossover events. By using five selective agar plates, we also showed thymidine auxotrophy of ITD and ITDAD and D-alanine auxotrophy of IAD and ITDAD. In M17G and skim milk (SYG) media, the growth of the three recombinants was limited. In MRS media, the growth of IAD and ITDAD was limited, but ITD showed a normal growth pattern as compared with the wild-type strain (WT). All the recombinants showed maximal BmpB expression at an early stationary phase when they were cultivated in M17G supplemented with thymidine and D-alanine. These results suggest that auxotrophic recombinant L. lactis expressing a heterologous protein could be generated to reduce the ecological risks of a recombinant L. lactis.
authors.affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
authors.forename: Eun Bae
authors.initials: EB
authors.lastname: Kim
authors.forename: Jee Soo
authors.initials: JS
authors.lastname: Son
authors.forename: Qian Kun
authors.initials: QK
authors.lastname: Zhang
authors.forename: Nam Kyung
authors.initials: NK
authors.lastname: Lee
authors.forename: Sung Hee
authors.initials: SH
authors.lastname: Kim
authors.forename: Jin Huk
authors.initials: JH
authors.lastname: Choi
authors.forename: Sang Kee
authors.initials: SK
authors.lastname: Kang
authors.forename: Yun Jaie
authors.initials: YJ
authors.lastname: Choi
mesh_terms: D000409:Alanine / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003470:Culture Media; D015870:Gene Expression; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D000409:Alanine; D013936:Thymidine