Generation and characterization of thymidine/D-alanine auxotrophic recombinant Lactococcus lactis subsp. lactis IL1403 expressing BmpB.
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.
Kim
Eun Bae
EB
Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
Son
Jee Soo
JS
Zhang
Qian Kun
QK
Lee
Nam Kyung
NK
Kim
Sung Hee
SH
Choi
Jin Huk
JH
Kang
Sang Kee
SK
Choi
Yun Jaie
YJ
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
D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D000409:Alanine; D013936:Thymidine