Preliminary Study On The Effect Of Protein Acetylation On Carbon Metabolism In Saccharomyces Cerecvisiae

The metabolic process of sugar(carbon source)is the most important metabolic process in organisms,which is often regulated strictly by signals.When the metabolic process is disordered,it often leads to disease or death of the cells in various degrees.Saccharomyces cerevisiae is a deep-seated eukaryotic model organism with food-grade safety,rich research tools,and relatively mature research techniques.In addition,the metabolism of S.cerevisiae is very promising,and it is very suitable as a cell factory for the production of various fermentation products.In recent decades,it has also made great achievements in the fields of metabolic engineering and synthetic biology.The sugar metabolism in S.cerevisiae.like most eukaiyotes,mainly includes the glycolytic pathway,the pentose phosphate pathway,the TCA cycle,the glyoxylate cycle,and the gluconeogenesis pathway.Under the condition that there is abundant fermentable carbon source(most typical:glucose)in the growth environment,the S.cerevisiae cells are in a strong glycolysis state,regardless of whether the oxygen supply is sufficient or not.Only a small amount of pyruvic acid flows into the TCA cycle for respiratory metabolism.most of which produces ethanol,and this effect is called the Crabtree effect,which has many similarities with the Warburg effect of cancer cells,namely rapid glycolysis metabolism.When glucose is depleted,yeast responds through cascaded signaling pathways,regulating gene expression and protein activity in major metabolic pathways from transcription,translation?post-translational protein modification.etc.,thereby precisely regulating metabolic networks to respiratory metabolism.Using ethanol.a non-fermentable carbon source accumulated during fermentation of glucose to perform "diauxic shift".Both the Crabtree effect and the"diauxic shift"switching process caused by the change of carbon source involve the most basic sugar metabolism regulation process of life activities,which has been long concerned by scientists.At present,the signaling systems and regulatory mechanisms involved in these processes are still at a limited cognitive level.The most well-known are the multiple regulatory pathways and feedback loops involved in transcriptional regulation and protein phosphorylation modification.Lysine acetylation is an important post-translational modification process in cells Over the years,researches have focused on the regulation of cell physiology inheritance by histone modifications.Recent studies have shown that protein acetylation plays an important role in regulating the metabolic processes of living organisms.Recent studies have also found that in addition to histones,a large number of metabolic enzyme proteins also have acetylation sites.Based on this,this paper explored the effects of acetylation on the carbon metabolism of S.cerevisiae from two aspects:the difference in acetylation of metabolic enzymes under different carbon sources and the effects of disturbance acctylation and deacetylation on carbon metabolism.In the first part of this paper,high-resolution mass spectrometry was first used to analyze the differences in the acetylation of S.cerevisiae under the conditions of fermentable carbon source glucose and non-fermentable carbon source ethanol.The results showed that under ethanol conditions,the acetylation level of glycolytic pathway,pentose phosphate pathway and ribosomal protein showed a downward trend:the level of protein acetylation in the Krebs cycle was up-regulated.Then.the metabolic pathway protein and the sugar signaling pathway protein with different acetvlation conditions under different carbon source conditions are selected,and the lysine at the acetylation site is mutated to arginine by the point mutation technique,so that the acetylation site becomes the non-acetylated state is formed.In turn,the effect of specific acetylation modification sites on protein activity was investigated by enzymatic assays in biochemical level.Compared with the control respectively,the activity of acetylation site mutant strain the pyruvate kinase(coding gene PYK1).malate dehydrogenase(coding gene MDH1),glucose-6-phosphate dehydrogenase(coding gene ZWF1)increased by 25.6%,41.4%,and 27.3%.respectively,while the fructose 1.6-bisphosphate aldolase(codins gene FBA1)acetylation site mutant strain decreased 19.9%.The change in the level of individual enzyme activity of the above metabolic enzyme does not affect the fermentation characteristics of the strain.However,the acetylation site of the alcohol dehydrogenase(coding gene ADH1)was mutated,the yield of the strain was significantly reduced when the strain was cultured under glucose condition,while the yield of ethanol increased and the acetic acid accumulated significantly,These results indicate that acetvlation affects metabolic enzyme activity in S.cerevisiae,and certain enzymes such as the Adh1 mutant,can significantly affect metabolism.The overall regulatory effects of multiple enzyme proteins which have differences in acetylation remain to be further demonstratedThe second part of this paper is to investigate the possible effects of acetylation on veast metabolism at other levels.We artificially disturb the acetylation process by knocking out the catalytic subunits or important constituent subunits of acetylase or deacetvlase,and study the effects of these perturbations on the metabolic characteristics of the strain.The results showed that the strain yield of knockout GCN5 and ADA2 was consistent with the control,and there was no promotion of glucose utilization,the yield of ethanol increased slightly.Knockout of NAT3 reduced the yield of strain and did not promote the utilization of glucose.There was a slight increase of ethanol:knockout of HOS2.HST2.SET3.RCO1,SDS3,and RPD3 did not accelerate the utilization of glucose,and the utilization and yield of ethanol did not increase significantly.Knocking out ARD1 and SAS3 accelerates the utilization of glucose in the fermentation metabolism stage,reduces the strain yield and increases the yield of ethanol.Knocking out HAT1 can accelerate the utilization of ethanol in the respiratory metabolism stage.HAT1 is also the known cytoplasm acetyltransferase in yeast(type B).These results indicate that acetylation is involved in cellular metabolic regulation of S.cerevisiae,but the specific mechanism remains to be further studied.Existing research of metabolome,transcriptome,proteome,phosphorylation have revealed the carbon metabolism of S.cerevisiae deeply.But there are still many unknown mechanisms for the regulation of metabolism.In recent years,it has been suggested that acetylation regulates cell physiological metabolism conserved from bacteria to humans.The research in this paperis a useful supplement to the study of protein acetylation-mediated metabolism regulation,and has broad implications for revealing metabolic mechanisms in the biological world and supporting evolutionary perspectives.