Study On The Mechanism Of Tolerance Response Of Candida Tropicalis To Phenol,an Inhibitor In Lignocellulose Hydrolysis

Lignocellulose biomass,as the most abundant and sustainable resource in the world,is extensively applied for fermentative production of biofuels,biochemicals,and food additives by microorganisms.But lignocellulose biomass needs to be converted into monosaccharides for fermentation.,among the numerous pretreatment methods,dilute sulfuric acid hydrolysis is the most simple,fast,and economic one.But,this method inevitably generates inhibitors,which could inhibit microbial cell growth and fermentation.The phenolics derived from lignocellulos have been identified as one of the most important categories of inhibitors.After pre-treatment of lignocellulose,the fermentation strain should be selected for subsequent enzymatic hydrolysis,and the study found that Candida tropicali showed high tolerance to a variety of inhibitors,and high temperature and salt resistance.A lot of research has been done on the degradation mechanism of phenol by C.tropicalis,but the tolerance mechanism of C.tropicalis to high concentrations of phenolic compounds is still unclear.Since the molecular structure and toxicity of phenol are similar to those of other phenolic compounds derived from lignin hydrolysis,we treated C.tropicalis SHC-03 with phenol as a representative substance for the phenolic compounds.In this paper,we observed the morphological changes of subcellular structures by fluorescence confocal microscopy and used the second generation of high-throughput sequencing technology to find out the key genes related to tolerance to phenol.The results of the trial are as follows:1.After treating cells with 0.5 g/L,1.0g/L and 2.0g/L phenol,the growth of C.tropicalis strain SHC-03 was inhibited,but fully recovered during the lag period.In addition,in YPD mediums with phenol,we measured the concentration of residual phenol using the 4-amino antibelin specular photometric method,showing no significant change in the concentration of phenol,indicating that the degradation mechanism of C.tropicalis to phenol was not activated,indicating that the cell-to-phenol tolerance mechanism plays a major role in maintaining cell vitality.2.According to the detection of intracellular reactive oxygen(ROS)content and the observation of subcellular structure,phenol can induce the accumulation of ROS,damage the structure of mitochondria and endoplasmic reticulum,but does not affect the structural stability of chromatin,and improves the resistance of cell walls to lysase,and low concentrations of phenol(0.5 g/L,1.0 g/L)affect the size and number of vacuoles in cell.3.According to transcriptome data,under the stress of phenol,there was no significant change in the expression level of the genes associated with phenol degradation pathways,which further confirmed that the degradation mechanism of C.tropicalis to phenol was not activated.On the contrary,the tolerance mechanism of C.tropicalis plays a major role.Among them,the expression level of the key enzyme genes for DNA synthesis/repair and the genes related to chromosomal transfer fidelity protein 18 were significantly up-regulatde,which could ensures genetic stability.Heat shock protein(HSP)-mediated protease degradation and autophagy activation,as well as autophagy formation associated genes also show up-regulatde expression,which helps maintain the stability of intracellular proteins and remove damaged proteins.And the up-regulatde expression of MDR/MXR transporter related genes in the efflux mechanism promotes the efflux of phenol and cytotoxic compounds in cells.However,the genes encoding the key enzymes of fatty acid degradation showed down-regulation,which resulted the accumulation of fatty acids to promote the regeneration and repair of organelles by membrane system.In addition,the genes related to the pathway of cell wall synthesis and degradation also show differential expression,which may lead to cell wall remodeling to reduce the introduction of phenol into cells.the Differential expression of all these genes together maintain the normal life activities of cells and increases the tolerance of C.tropicalis to phenol.In this paper,the damage mechanism of phenol to C.tropicalis strain SHC-03 cells,as well as the tolerance mechanism of C.tropicalis strain SHC-03 to phenol are explored,which lays the foundation for the later application of C.tropicalis to be used in lignocellulose fermentation process or to improve strain tolerance and construct new yeast engineering strains.