Studies On The Tolerance Mechanism Of Corynebacterium Glutamicum To Aromatic Compounds

Lignin,hemicellulose and cellulose are the major components of lignocellulose.It is the most abundant renewable resource on the earth and the most reliable source of raw materials for the biorefinery industry.Therefore,the efficient use of lignocellulosic substrates technology(such as co-fermentation of pentose and hexose sugars technology,cellulose degradation technology)has been the hotspot of biorefinery research.Through years of research,the above technical problems are gradually being solved.Improving its tolerance to growth inhibitors from pretreatment of lignocellulose biomass is becoming the next research hotspot in efficient use of lignocellulose.Among these inhibitors,phenolics(such as ferulic acid,vanillin,phenol,etc.)are one of the major inhibitors.Then improving the tolerance of industrial strains to these phenolics,and minimizing its impact on the industrial bacteria in the fermentation becomes an urgent problem to be solved.Corynebacterium glutamicum is an important industrial strain that plays an important role in the production of amino acids in microbial fermentation,especially in the production of glutamic acid in the industry and the production of monosodium glutamate has been widely used.Moreover,it is more talented than other typical industrial strains in degradation and tolerance to phenolic compounds from the pretreatment of lignocellulose biomass.Therefore,by taking Corynebacterium glutamicums as a research object,using microarray and molecular genetics methods,we aim to uncover its tolerance mechanism to ferulic acid,vanillin and phenol.The main results are as follows:1.The tolerance mechanism of C.glutamicum to ferulic acid is summarized as follows:(1)C.glutamicum can degrade ferulic acid(degradation-related genes: phdA-E,vanAB,pcaGH were up-regulated),which reduces the toxic effect to cells by reducing the concentration of ferulic acid in the growth environment.(2)The complex structure of cell envelope in C.glutamicum has greater protective effect than other bacteria,and several genes involved in the synthesis of cell wall and membrane protein also have differentially expressed to adapt to ferulic acid stress.(3)The growth of C.glutamicum cells will slow down(certain replication,transcription and translation of genes were down-regulated),which can reduce damage to DNA,RNA and protein under ferulic acid stress.(4)C.glutamicum cells can activate a variety of defense and repair mechanisms to deal with the effects of ferulic acid(induction response,production of Clp family proteins to degrade damaged proteins and protein modifcations,etc.).Further studies have shown that sigE and katA mutants are more sensitive to ferulic acid stress.2.The tolerance mechanism of C.glutamicum to vanillin is summarized as follows:(1)C.glutamicum can degrade vanillin(degradation-related genes: vanAB,pcaBCH were up-regulated),which reduces the concentration of vanIllin surrounding cells.(2)The C.glutamicum cell envelope,which has complex structures,has a greater protective effect than other microbes.(3)Under the condition of vanillin stress,some differentially expressed genes related to stress response can reduce the damage to C.glutamicum cells.(4)Ribosome/translation and protein secretion genes were differentially expressed to cope with the vanillin stress.sigH and msrA mutants were more sensitive to vanillin stress.3.The tolerance mechanism of C.glutamicum to phenol is summarized as follows:(1)C.glutamicum can degrade phenol(degradation-related genes: phe,pcaDF were up-regulated),which reduces the concentration of phenol around the cells.(2)C.glutamicum reduces the damage to the biomacromolecules by reducing the rate of cell growth and metabolism(several replication,translation,and transcription-related genes were down-regulated).(3)The protective effect of the cell envelope of C.glutamicum is much stronger than others.(4)C.glutamicum can induce several protective and repair mechanisms to accommodate phenol stress(induction heat shock reactions,producing chaperone proteins to help protein folding,etc.).Further experimental results indicate that groEL mutant is more sensitive to phenol stress.4.By analyzing the microarray data of C.glutamicum response to ferulic acid,vanillin and phenol,the differentially expressed genes in all three microarray data were analyzed.The number of differentially expressed genes was 140(73 up-regulated,67 down-regulated).Based on this,we obtained the interaction network of differentially expressed genes by using the analysis of STRING,and then analyzed five important node proteins(NCgl2553,NCgl1897,NCgl2586,NCgl0802,NCgl0882).And then through the metabolic pathway and other methods for further analysis,we found that differentially expressed genes were involved in the pathways of utilization of carbon sources and the cell membrane-related genes.This suggests that C.glutamicum copes with the effects of toxic phenolic aromatic compounds(ferulic acid,vanillin and phenol)by adjusting the carbon metabolism mechanism and the cell membrane related genes.