Nickel Tolerance Mechanism Of Saccharomyces Cerevisiae H4K5R Based On Metabolomics

Nickel（Ni）is one of the heavy metal contaminants widely exposed to humans.In nature,many microorganisms resist nickel toxicity through various mechanisms,including anti-organic acids,alcohols,and extracellular polymers.Many scholars have studied the mechanism of nickel resistance in microorganisms based on transcriptome,but relatively few have studied it at the metabolic level.Through previous work by our group,the nickel-tolerant strain H4K5R has been selected,however the mechanism of why it is nickel-tolerant is not clear.In this study,we analyzed the nickel tolerance mechanism of S.cerevisiae H4K5R based on histone site-directed mutant strain H4K5R（Lys replaced by Arg at position 5 of histone H4 to simulate the intracellular deacetylation state）,combined with metabolomics and cell wall integrity pathways under nickel stress.Main study results are as follows:1.With the increase of nickel stress concentration,the intracellular ROS content of both wild strain BY4741 and mutant strain H4K5R increased,but the intracellular ROS content of mutant strain H4K5R was significantly lower than that of wild type;the reduced glutathione GSH content decreased and the SOD content increased in the antioxidant system of wild strain BY4741 under nickel stress,and the reduced glutathione GSH content and SOD content were at a low level in mutant strain H4K5R.Compared with the wild strain,the mutant strain was exposed to less oxidative stress under nickel stress,the mutant strain had oxidative resistance.2.PCA modeling was used to analyze the metabolome data.A total of 285 differential metabolites were identified between the mutant H4K5R and the wild strain BY4741,mainly including amino acids,sugars,and nucleotides,of which 5-L-glutamyl-L-alanine was the glutathione substance,and the increase in the content of 5-L-glutamyl-L-alanine in the mutant H4K5R may be one of the reasons for its strong antioxidant properties.3.The metabolites significantly different between the mutant strain H4K5R under nickel stress and the wild strain BY4741 include amino acids,sugars,and nucleotides.In the wild strain BY4741 under nickel stress,the content of D-mannose-6-phosphate,the main component of mannan in the cell wall,increased,and the contents of hydrogen phosphate and inosine decreased.It was speculated that the wild strain responded to nickel stress by increasing the content of D-mannose-6-phosphate,an important component of mannan,to stabilize the cell wall,and then reducing energy consumption;the mutant strain H4K5R had better cell signal transduction system and sufficient energy supply by increasing the content of phosphatidylethanolamine,one of the important members of phospholipids,and citric acid,an intermediate product of the clostridium cycle,under nickel stress,so that the mutant strain H4K5R had nickel tolerance.4.Through KEGG pathway enrichment analysis,the metabolic pathways enriched for differential metabolites between mutant H4K5R and wild strain BY4741 were arginine biosynthesis,alanine,aspartate and glutamate metabolism,and tryptophan metabolism,respectively;under nickel stress,the enriched metabolic pathways included yeast cell cycle,tricarboxylic acid cycle,biosynthesis of pantothenic acid and Co A,and histidine metabolism in addition to alanine,aspartate,glutamate metabolism,and arginine biosynthesis.5.The cell wall integrity pathway was activated in wild strain BY4741 under 5 m M nickel stress,the expression of mannan and glucan regulatory genes Mnn9 was significantly up-regulated 3.13-fold,the expression of Fks1 was significantly up-regulated1.49-fold,and the contents of mannan andβ-glucan were also increased,indicating that the wild strain responded to nickel stress by replenishing the cell wall components at this time;the cell wall integrity pathway was slightly activated in mutant strain H4K5R under 5 m M nickel stress,although the expression of Mnn9 and Fks1 was up-regulated,the content ofβ-glucan was increased but the increase was small compared with the wild strain,while the change in mannan content was not significant,indicating that the mutant strain H4K5R had nickel tolerance by activating the mechanism of CWI pathway to regulate the cell wall components under 5 m M nickel stress.In summary,the mutant H4K5R had oxidative resistance compared with the wild strain BY4741,and under 5 m M Ni Cl₂stress,the mutant had nickel tolerance by regulating its own basal metabolites,as well as a more stable cell wall structure.