earticle

영어

Optimal microbial strains for bioethanol fermentation must utilize various carbon sources and withstand stressful conditions such as high temperature, high osmotic pressure, high concentration of ethanol, and, in some cases, extreme pH. We have developed a method in microbial cells that uses combinatorial libraries of zinc finger–containing, artificial
transcription factors to induce stress resistance and to identify genes involved in stress resistance. By linking multiple zinc finger domains together, we constructed more than 300,000 zinc finger proteins with varied DNA-binding specificities. These zinc finger proteins were fused to appropriate effector domains to produce libraries of transcription factors. Zinc finger transcriptional regulatory proteins, then, were expressed individually in yeast or in E. coli, and the transformed cells were screened for organic solvent tolerance and thermotolerance. Combining chromatin immunoprecipitation experiments and in silico prediction of target DNA
binding sequences for the artificial transcription factors, we identified novel genes associated with the stress resistance in E. coli. Our results show that randomized libraries of artificial transcription factors are powerful tools for functional genomic studies and for microbial engineering.