Mechanism Of ATG Gene Deletion Improving Ethanol Tolerance In Saccharomyces Cerevisiae

S.cerevisiae is not only widely used as a eukaryotic model organism in basic research such as genetics and biochemistry,but also a significant producer of alcoholic beverages and ethanol fuels.Although the strains produced by fermentation have good ethanol tolerance,the continuous increase of ethanol concentration will inhibit cell growth and vitality,which has become an important limiting factor to improve the fermentation capacity.At present,the tolerance mechanism of yeast to ethanol stress is not completely clear.To further understand the related mechanism,this thesis carried out the following exploration:Firstly,we detected the cytotoxicity of ethanol stress on the wild-type BY4741 strain.The results showed that the inhibition of cell growth and division was increased with treatment concentration.Meanwhile,the accumulation of TBARS induced by a high ethanol concentration was much higher than that in the control group.PI staining showed that the integrity of the cell membrane was damaged under ethanol stress,and the number of cell death was significantly higher than that of the control group.It was reported that reactive oxygen species is an important factor of ethanol stress cells in the process of fermentation.We used fluorescent probe DCFH-Da to detect intracellular ROS levels and found that intracellular ROS levels were increased slightly under ethanol stress.There was no significant difference compared with the control group.Exogenous antioxidant Vc could not block ethanol-induced cytotoxicity,which could be inferred that ROS was not the main factor of ethanol-induced cell toxicity.The ethanol stress-sensitive phenotype of the strain with oxidative stress-related gene deletion was detected.It was found that YAP1 was highly sensitive to 2 m M H₂O₂ and insensitive to 10%ethanol,which again indicated that reactive oxygen species were not the main reason for ethanol inhibition of cell growth.Ethanol stress was associated with autophagy.Our results showed that the mutation of ATG9,a key autophagy gene,was tolerant to ethanol stress.Further detection showed that the expression levels of ATG8 m RNA and protein were significantly increased under ethanol stress,and antioxidant Vc could not significantly reduce the intensity of autophagy,indicating that autophagy played an important regulatory role in ethanol inhibiting the growth of yeast cells.Subsequently,we detected the growth status of autophagy defects atg1Δ,atg9Δand atg10Δunder ethanol stress.It was found that ATG mutations had apparent ethanol resistance.The addition of autophagy inducer rapamycin Rap significantly inhibited the growth of ATG mutant strains.However,the addition of TOR activator KIC（4-methyl-2-isovalerate）did not significantly improve the tolerance of wild-type BY4741 to 10%ethanol and reduce the relative growth advantage of the mutant as expected.TORC is the central regulator of cell nutrition metabolism and plays a vital role in regulating intracellular carbon,nitrogen metabolism and autophagy.Ethanol is the intermediate product of glucose metabolism.Therefore,it is speculated that the TORC-mediated glucose metabolism pathway is involved in ethanol resistance of ATG mutants.Herein,we used galactose,glycerol and ethanol to replace glucose in the medium and detected the genetic activity of ATG-deficient strains under ethanol stress.It was found that autophagy-deficient strains showed similar growth advantages in galactose,glycerol and 2%glucose medium but had no growth advantages in ethanol medium.Because the metabolic utilization of galactose and glycerol goes through glycolysis pathway,while ethanol does not,it is speculated that ethanol tolerance of ATG-deficient strains is related to the glycolysis process.2-deoxyglucose（2-DG）and sodium citrate were added to the ethanol stress medium to inhibit the glycolysis pathway.It was found that the ethanol tolerance of wild type BY4741 strain was improved.At the same time,the ATG mutations atg1Δ,atg9Δand atg10Δgrew worse than the wild type.It could be concluded that the tolerance of autophagy-deficient strains to ethanol depends to a certain extent on the integrity of the glycolytic pathway.It was indicated that the deletion of crucial enzyme genes（tdh1Δ,tdh2Δ,tdh3Δ,pfk1Δand hxk2Δ）encoding the glycolytic pathway will show better growth over the wild-type strain under ethanol stress.The results confirmed that the interruption of the glycolysis process from the genetic pathway could improve the ethanol tolerance of mutant cells.However,the activation of the tricarboxylic acid（TCA）cycle by adding sodium pyruvate did not change the growth inhibition of ethanol.Thus,the ethanol tolerance process of autophagy-deficient strains did not depend on TCA cycle.Meanwhile,the detection of autophagy intensity of pfk1Δalso showed that the autophagy level in cells remained low after the inhibition of the glycolysis pathway,which was more conducive to the tolerance of Saccharomyces cerevisiae cells to ethanol.PFK1 was knocked out based on atg9Δ,and the atg9Δ/pfk1Δdouble deletion mutant was constructed.It was found that the growth advantage of the atg9Δdeficient strain was eliminated.Again,ATG mutation interrupted autophagy induced by glycolysis pathway,so as to improve ethanol tolerance in Saccharomyces cerevisiae.In summary,ethanol stress can cause oxidative damage to yeast cells,but it is not the dominant factor of its growth inhibition.Excessive activation of autophagy can inhibit yeast growth under ethanol stress,and ATG mutation can interrupt the autophagy process induced by the glycolysis pathway to improve ethanol tolerance of Saccharomyces cerevisiae.This study expands the understanding of cell tolerance mechanism under ethanol stress and also provides a new solution for improving ethanol fermentation capacity.