2002 Fiscal Year Final Research Report Summary
Improvement of organic solvent tolerance level by directed evolution of AcrAB-TolC efflux pump in Escherichia coli
| Project/Area Number |
12460042
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
応用微生物学・応用生物化学
|
|---|
| Research Institution | Tokyo Institute of Technology |
|---|
Principal Investigator |
DOUKYU Noriyuki Tokyo Institute of Technology, Bioinformation, Assistant Prof., 大学院・生命理工学研究科, 助手 (60302957)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TSUKAGOSHI Norihiko KUBOTA Corporation, 研究員
|
|---|
| Project Period (FY) |
2000 – 2002
|
| Keywords | Escherichia coli / organic solvent / organic solvent tolerant / AcrAB-TolC efflux pump |
|---|
| Research Abstract |
Several physiological and biochemical approaches have been applied in an effort to elucidate the mechanisms of microbial resistance to solvents. In recent years, it has been demonstrated that energy-dependent efflux is involved in organic solvent tolerance in gram-negative bacteria. Cloned genes coding for components of efflux pumps belonging to the resistance/nodulation/cell division (RND) family have been shown to serve to maintain solvent resistance in certain bacteria. Genetic evidence suggests that the AcrAB-TolC efflux pump, a member of the RND family, is involved in solvent resistance of E. coli cells. We showed that the AcrAB-TolC pump actually reduces the intracellular solvent concentration in E.coli cells exposed to a solvent. In this study, we estimated whether the organic solvent level in E.coli improves by using in vitro directed evolution. The family shuffling method and error prone PCR were applied for in vitro directed evolution to construct a mutant library of acrB genes. The organic solvent tolerance levels of E.coli strains harboring mutant acrB genes were lowered. This result shows that mutant AcrB, of which efflux function was changed, was constructed. We searched other efflux pump that has higher activity than that of AcrAB-TolC pumps from AcrAB homologues. As the results, it was shown that AcrAB-TalC pump shows higher activity than other AcrAB homologues tested.
|
