Study On Tolerance Mechanism Of Arthrobacter Simplex Under Ethanol Stress

Arthrobacter simplex,a producing-strain with steroids C1,2 dehydrogenationability has been widely used in the steroid industry.The addition of organic solvents(such as ethanol)can help better dissolution of hydrophobic substrates.In this study,Arthrobacter simplex TCCC 11037 was selected as a research model,thesis analyzed the survival ratio,DNA damage,relevant metabolite content(ROS levels)and the change of expression level of relative resistant genes to explore the resistant mechanism of the Arthrobacter simplex resistant to ethanol.These achievements may provide a theoretical guidance for creating a steroid C1,2 dehydrogenation with high organic solvent tolerance.dnaK,groEL,recA,uvrD,katG,SOD were respectively amplified by PCR using A.simplex TCCC 11037 genome as a template.The nucleotide sequence was obtained.The homology analysis and phylogenetic tree were further carried out.Bioinformatics analysis of target proteins were discussed,This genes were expressed in E.coli BL21(DE3).By analyzing the different ethanol treatment on the survival ratio,DNA damage,relevant metabolite content(ROS levels)and the expression level of relative resistant genes,thess explored the molecular mechanism of Arthrobacter simplex resistant to ethanol.The cells cultured with different concentration of ethanol for 3 h.It was found that favourable growth property were observed when ethanol concentration is no more than 4%,while the survival ratio decreased obviously when we continue to increase the concentration of ethanol.When the ethanol concentration was 8%,the survival ratio decreased only to 15.12%,the damage rate of DNA and ROS accumulation in bacterial cells was significantly increased by 7.6%and 63%.The results showed that the ROS accumulation are important causes of DNA damage,thereby causing decreased cell viability under the stress of ethanol.When ethanol was 8%,catalase enzyme activity and total SOD activity were improved by 66%and 29%,According to the further analysis of the expression levels of relative genes,it was reported that the relative expression levels of DNA repair gene(recA and urvD)were increased by 2.59 fold and 2.57 fold,and relative expression levels of oxidative stress-related genes(katG)went up to 2.54 fold,Furthermore,the relative expression levels of heat shock protein(dnaK and groEL)increased by 10.41 fold and 6.31 fold respectively.When the cells were cultured with different concentration of ethanol for 12 h.The results was same as the different concentration of ethanol for 3 h.The results showed that the ROS accumulation are important causes of DNA damage,thereby causing decreased cell viability under the stress of ethanol.Thesis researched the effects of different resistant conditions of recombinant Bseherichia coli(e.g.50℃,methanol,sorbitol and pH=3).The results demonstrated that heterologous expression of GroEL gene in Arthrobacter simplex could significantly improved the survive ratio of Escherichia coli in the resistant conditions of high temperature,methanol,hypertonic and acid stress,improved the livability of Escherichia coli in the resistant conditions of methanol,hypertonic and acid stress.