The Study On Physiological Functions Of Mycothiol In Corynebacterium Glutamicum

Corynebacterium glutamicum is used for the large-scale industrial fermentation ofamino acids, and the production of nucleotide, and vitamins. Metabolic engineering studies inrecent years have shown that C. glutamicum is also capable of producing a variety of manybio-based commercially interesting compounds, e.g. organic acids, diamines, biofuels, orhigh value proteins. However, there are all kinds of stressful conditions during fermentation.In order to cope with the stressful fermentations, high-resistant and industrial performancerobust microorganisms are needed. The above sresses have same effect-oxidative stresses,which inactivates biomacromolecules by forming ROS. Two sets of systems are evolved tocope with oxidative stresses: enzymes and nonenzymes. The latter refers mainly to lowmolecular weight thiol, including Cys、CoA、vitamin C/E、GSH. Among them, the functionof GSH is very clear. More importantly, GSH metabolism pathway has been successfullyapplied in improving engineering of physiological functionality and biological robustnessunder stressful conditions. Actinomycete, however, don not produce GSH, instead theyproduce MSH, wihich is essential to Mycobacterium tuberculosis growth. Many studies haveshown that MSH plays very broad and essential roles in the detoxification of oxidants, toxins,antibiotics, and in the degradation of aromatic compounds. The study of MSH in recent yearsmainly focused on whether it can be promising drug target, which ignores the study onphysiological functions of MSH. Today, very little is known about the physiological functionsin C. glutamicum, and whether the antioxidantive perpoty of MSH can be used to improve therobustness in C. glutamicum under stress. To this ends, we intensively investigated thephysiological functions and the internal mechanisms of MSH by using C. glutamicum as amodel organism. The main results are as follows:1．We investigated the roles of MSH takes part in detoxification of alkylating agents、oxidants、antibiotics、heavy metals、pesticide、acid、ethanol and aromatic compounds in C.glutamicum by using wild type, MSH-deficient mutants and corresponding complementarystrains. We found that all mutants were significantly more sensitive to toxins（Methylglyoxal、CDNB、mBBr、Iodoacetamide）、oxidants（H2O2、Diamide）、 antibiotics（erythromycin、 streptomycin、neomycin、gentamycin、spectinomycin）、resorcinol、naphthalene、glyphosate、ethanol and heavy metals (CdCl2、ZnCl2、CoCl2、MnCl2、CuSO4）than wild type, these datasuggest that MSH contributes to detoxication of above stresses in C. glutamicum, andrevealed the physiological functions of MSH.2．We revealed the antioxidative mechanism of MSH using acid strss as a model. Wefound that the survival of MSH-deficient mutants was fallen sharply under acid stress. MSHdeficiency resulted in ROS level increased, pHi decreased and intracellular methionine poolexhausted in MSH mutants under acid stress. There are three mechanisms in resistance ofMSH to acid stress:(1) maintaining pHihomeostasis;(2）scanvenging ROS;(3) protectingmethionine synthesis from acid stress by S-mycothiolation modification of MetE.3．MSH has many anti-stress properties in C. glutamicum under stress, which facesfrequently in fermentation of bio-based material. So, MSH over-production may be aprosming way to improve the industrial property of C. glutamicum. Then, we overexpressedMshA in C. glutamicum using genetic engineering, and found that intracellular MSH wasincreased markly in WT(pXMJ19-mshA) compared to WT(pXMJ19). Survival rates increasedby43.8,39.4,90,77,131,87.3,51.9,46.9,57.4,85.2and32.5%as compared toWT(pXMJ19) in the presence of H2O2(40mM), methylglyoxal (5.8mM), erythromycin(0.08mg/ml), streptomycin (0.005mg/ml), Cd2+(0.01mM), Mn2+(2mM), formic acid(0.05%), acetic acid (0.15%), levulinic acid (0.25%), furfural (7.2mM), and ethanol (10%),respectively. ROS levels in WT (pXMJ19-mshA) also decreased sharply compared toWT(pXMJ19) in the presence of above stresses. In the presence of H2O2(33mM), acetic acid(0.05%), furfural (16.7mM), ethanol (6.5%), CdCl2(15μM), the amino acid production ofGlu, Cys, Val, Met, Ile, Leu, Try, Phe and His in WT (pXMJ19-mshA) compared to WT(pXMJ19) increased from10.2%to100.3%. MSH, therefore, may significantly improve therobustness of C. glutamicum under stress, and provide a new way for engineering ofphysiological functionality in C. glutamicum.