Mechanisms Of Mycothiol Disulfide Reductase In Resistance To Multiple Environmental Stresses In Corynebacterium Glutamicum

As a very important industrial microorganism,Corynebacterium glutamicum will inevitably meet a variety of environmental stimulus in the fermentation process of biological products,which could cause an adverse effect on its fermentative capability.Also,these adverse environmental stimulus induces ROS production which can destruct redox homeostasis of cells,and then leads to damage of biomacromolecules which causes cell death.Therefore,improving robustness of C.glutamicum on unfavorable environment has become the focus of lots of research on C.glutamicum.To cope with the adverse effects of ROS,microbiology has evolved enzymatic antioxidant systems(such as Cat,SOD,Trx,Mtr),and non-enzymatic antioxidant systems.Mycothiol disulfide reductase(Mtr)can reduces disulfide form mycothiol disulfide(MSSM)to mycothiol(MSH),thus maintaining intracellular redox homeostasis.However,we know very little about the physiological roles of Mtr in C.glutamicum.Therefore,in this study we concentrate on mycothiol disulfide reductase(Mtr)in C.glutamicum,in order to revealing the physiological roles of Mtr.The results are as follows:1.When we attempt to delete the mtr gene in C.glutamicum,8000 target strains would recover to wild type,suggesting that Mtr is essential in C.glutamicum.2.In this study,a recombinant plasmid was constructed to overexpress Mtr in C.glutamicum using the expression vector pXMJ19-His6.Mtr-overexpressing C.glutamicum cells showed increased tolerance to ROS induced by oxidants,bactericidal antibiotics,alkylating agents,and heavy metals.3.By comparing the ROS levels and the protein carbonyl contents of Mtr-overexpressing C.glutamicum cells and wild type in various stressors,we found that Mtr overexpression could decrease the ROS level and the protein carbonyl level on oxidative stress.4.By comparing the reversible protein thiols of Mtr-overexpressing C.glutamicum cells and wild type in various stressors,we found that Mtr overexpression could increase the levelof reversible protein thiols on oxidative stress,in order to protecting protein against being oxidized.By comparing MSH:MSSM ratio in Mtr-overexpressing C.glutamicum cells and wild type in various stressors,we found that Mtr overexpression could largely recover the MSH:MSSM redox ratio and maintain cellular redox homeostasis.5.By comparing the activity and expression level of some antioxidant enzymes in Mtr-overexpressing C.glutamicum cells and wild type in various stressors,we found that Mtr overexpression could increase the activity and expression level of some antioxidant enzymes which leading to improve the stress tolerance of C.glutamicum.6.The results showed that mtr expression responds to multiple stress inducers at the transcriptional level and SigH can promote mtr expression on both transcriptional level and post-transcriptional level through ?-galactosidase activity assay and Real-Time fluorescence quantitative PCR(qRT-PCR)experiments.The electrophoretic mobility shift assay(EMSA)showed that SigH can directly and specificly bind to mtr promoter,In summary,in this research,we found that Mtr participates in protection against various oxidants in C.glutamicum through decreasing the ROS level directly or indirectly,increasing the activity and expression level of some antioxidant enzymes,increasing the level of reversible protein thiols and largely recovering the MSH:MSSM redox ratio.The results were important to enhance the resistance of C.glutamicum to adverse stress.