Study On The Mechanism Of Corynebacterium Glutamicum GprA Protein Against Phenolic Stress

Corynebacterium glutamate belongs to gram-positive bacteria,as one of the model strains to study the molecular mechanism,it is an important industrial fermentation strain,which can ferment and produce a variety of amino acids and small molecule compounds.Because of its good phenolic degradation and tolerance ability,it is one of the most promising chassis microorganisms for lignocellulose biotransformation.According to the previous study,it was found that GprA,a putative protein of unknown function of Corynebacterium glutamate,plays an important role in the stress of environmental factors such as anti-phenols,and the target protein mycobacterium acyltransferase A（MytA）interacting with GprA was preliminarily screened.Based on the verification of the interaction between GprA and MytA,this paper further studied the effect of mytA deletion on the anti-phenolic and other environmental stress ability of Corynebacterium glutamate,verified whether MytA participated in the anti-phenolic stress function of GprA,and elucidated the influence of GprA on the activity of MytA esterases in vitro and the key interaction sites,thereby revealing the molecular mechanism of GprA’s mediation of anti-phenolic stress by Corynebacterium glutamate through MytA.The results will help us further understand the environmental adaptation mechanism of Corynebacterium glutamate,and also provide ideas for us to further modify genetic engineering.The results of the study are as follows:1、q RT-PCR experiments,growth stress experiments and survival rate experiments showed that MytA was involved in the resistance of Corynebacterium glutamate to phenols,peroxides,heat,ethanol and acid stresses.2、ΔmytA mutants and complementary strains were constructed,and survival experiments showed that MytA participated in GprA-mediated anti-phenolic stress.Through fluoroscopic electron microscopy and thin layer chromatography analysis,it was found that similar toΔmytA mutant,gprA deletion led to defects in the cell envelope structure of Corynebacterium glutamate,and the content ratio of the cell envelope lipid trehalose monomycoate（TMM）/trehalose dimycoate（TDM）was increased.3、Molecular docking experiments,bacterial double heterogeneous experiments and in vivo co-expression experiments showed that there was interaction between GprA and the N-terminal catalytic domain of MytA.4、Related clones were constructed,and GST-GprA protein,His₆-MytA,His₆-MytA_44-406 protein and GST-GprA_mut tripod protein were purified,It was found that GprA protein could significantly enhance the N-terminal esterase activity of MytA,and GST-GprA_mut could not promote the in vitro esterase activity of His₆-MytA,indicating that the 24th Asn residue,the27th Lys residue and the 28th Val residue were the key binding sites of GprA to promote MytA in vitro esterase activity.