We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Generation and Characterization of Thymidine/ d-Alanine Auxotrophic Recombinant Lactococcus lactis subsp. lactis IL1403 Expressing BmpB.
- Authors
Eun Bae Kim; Jee Soo Son; Qian Kun Zhang; Nam Kyung Lee; Sung Hee Kim; Jin Huk Choi; Sang Kee Kang; Yun Jaie Choi
- 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.
- Subjects
THYMIDINE; PYRIMIDINE nucleotides; ALANINE metabolism; RECOMBINANT microorganisms; MICROBIAL genetic engineering; RECOMBINANT viruses
- Publication
Current Microbiology, 2010, Vol 61, Issue 1, p29
- ISSN
0343-8651
- Publication type
Article
- DOI
10.1007/s00284-009-9572-y