Butanol-tolerant Global Transcriptional Factor Mutant In Escherichia Coli And Its Mechanism Study

Microorganisms play important roles in industrial production of medicine, agriculture and fine chemicals and so on. However, organic solvents survived in the environment are toxic to the microorganisms by destroying the physiological functions. Hence it is especially important to obtain organic-solvent tolerance(OST) microorganisms in industrial applications.In this study, global transcription machinery engineering(g TME) was used to improve the OST of E. coli JM109. The mutant library of σ70 encoded by rpo D was screened under butanol pressure, and finally one mutant of E. coli JM109(named B8) harboring σ70was identified with capability of tolerating 2.0%(v/v) butanol. Then total transcriptional level of mutant B8 and E. coli JM109 was studied by DNA microarray analysis. The results show that there are 329 genes exhibiting over 2-fold expression difference between mutant B8 and JM109 under 0.8%(v/v) butanol, which included 197 up-regulated genes and 132down-regulated genes. These genes involved in glucose metabolism, amino acid, membrane proteins, nucleic acid and efflux pumps. In this study, three up-regulated gene(yei C, yei N,Ecs0572) was silenced by as RNA, and 2 down-regulated gene(yrb L, yhc N) were knocked out to evaluate their OST-related properties in E. coli. Our results indicate that yei C and yei N play important roles in improving OST of E. coli. Genes yei C and yei N are involved in the synthesis pathway of pseudouridine, the most abundant modified nucleoside presented in RNA. Gene yrb L was found to be repressed in the presence of high concentrations of extracellular Mg2+, which is known to enhance the stability of the cell membrane. The function of gene can regulate gene fab R. Gene fab R which encodes DNA-binding transcriptional repressor inhibits the synthesis of membrane proteins. Gene deletion of yhc N inhibits the expression of fab R and results in significantly elevated levels of unsaturated fatty acids.This work provides experimental data and molecular basis for constructing OST bacteria for industrial applications.