The Regulation Function Of Transcription Factor Dal80p In The Nitrogen Metabolism Of Saccharomyces Cerevisiae Was Investigated Based On Comparative Transcriptomics And ChIp-Seq

Ethyl carbamate is one of the most widely distributed grade 2A carcinogens in fermented food,especially in fermented drinks.The main reason for its formation is the selectivity and underutilization of nitrogen source caused by the nitrogen catabolite repression effect of Saccharomyces cerevisiae.Deeply understanding of the nitrogen catabolite regulation mechanism of Saccharomyces cerevisiae and metabolic engineering modification of fermentation strains are the most promising ways to solve the formation of ethyl carbamate in fermented drinks.Dal80 is one of the negative transcriptional regulators of nitrogen repression in Saccharomyces cerevisiae incompletely understand.In this study,Saccharomyces cerevisiae BY4741,dal80 knockout and overexpression strains were used as the experimental strains to systematically study the function of Dal80 p in the regulation of nitrogen metabolism by using RNA-seq,ChIP-Seq,conventional physiological and biochemical evaluation methods.1.Research of Dal80 p influence strain nitrogen source utilization and growth were implemented and results shown: Dal80 p pose no significant effect on strain growth,but dal80 expression can significantly inhibit the total amino acids and serine,phenylalanine,tyrosine,alanine,glycine,arginine,leucine,methionine,threonine,histidine and cysteine amino acids utilization,increase urea accumulation of arginine degradation under poor nitrogen source conditions。2.Experiments of prefer nitrogen source inhibit arginine and urea utilization were carried out and results revealed : prefer nitrogen source inhibited arginine using more significant than urea.The deletion of dal80 is advantageous to strain utilize arginine under prefer nitrogen source and reduce intracellular accumulation of urea produced in arginine degradation process.On the contrary,dal80 overexpression increases the accumulation of urea both intracellular and extracellular.Glutamine is the preferred nitrogen source with the most significant inhibition effect on arginine utilization,and the increase of its concentration will reduce the activity of arginine and urease,while the deficiency of dal80 can help alleviate the decrease of enzyme activity.3.The role of dal80 p in the inhibition of arginine utilization induced by low XIV concentration glutamine were researched by means of RNA-seq.It was found that the deletion of dal80 up-regulated the expression of genes related amino acid metabolism,cell cycle,glycolysis,MAPK signaling pathway,while the overexpression was opposite.Among them,amino acid metabolism related genes are mainly amino acid transport and degradation related enzymes.RT-q PCR verified that the deletion of dal80 can significantly down-regulate the glutamate synthase gene gdh1 and argininase gene car1,and up-regulate the expression of the glutamate degraded enzyme gdh2,glutamate synthase gdh3,urease dur1,2 and arginine transporter alp1,but has no significant effect on the expression of arginine transporter gene gap1.4.The role of dal80 p in the inhibition of arginine utilization induced by high concentration glutamine were researched by means of RNA-seq.Results shown that the increase of glutamine concentration would inhibit the expression of a large number of amino acid transport and metabolism-related enzymes genes in BY4741,but this inhibition would be removed or even reversed with the deletion of dal80.In addition,the overexpression of dal80 also showed significant down-regulation on expression of ribosomal metabolism-related genes.In addition,dal80 overexpression also significantly inhibited the expression of ribosomal metabolism-related genes.RT-PCR verification results show that deletion of dal80 can up-regulation the expression of arginase car2,urease dur1,2,arginine transporters gap1,alp1 and amino acid permease agp1,agp2 and arginine synthesis related genes arg1 and arg4,while inhibit the expression of arginine enzyme car1.Meanwhile,the expression of glutamine synthetase gln1,glutamate dehydrogenase gdh1,gdh3 were increased but the expression of glutamate dehydrogenase gdh2 was decreased.This may indicate that the absence of dal80 helps to alleviate the inhibition of glutamine on its utilization by enhancing the transport of arginine and degradation of decomposition products.5.The potential targets of Dal80 p were explored by means of ChIP-Seq and results consistent with RNA-seq: Da80 p may affect on genes related cell cycle,cell division,amino acid synthesis,MAPK signaling pathways,ribosomes and other metabolic pathways.Among all genes regulated,amino acid synthesis mainly focuses on enzymes related transport and degradation of amino acids,especially associated with arginine and glutamine.In addition,we also found that Dal80 p may interact with other proteins to complete transcriptional regulation cooperatively,and may bind at the transcriptional termination sites in addition to the transcriptional initiation sites.Arginine metabolism related genes captured in Peak concentration shown highly consistent with amino acid metabolism related genes up-regulated by dal80 deletion and mainly belong to enzymes act on arginine transport,synthesis,and metabolites.It is suggests that dal80 may inhibit arginine transport,synthesis conditions,decomposition of the product to repress the utilization of arginine under glutamine conditions.In addition,RT-q PCR results showed that dal80 deletion up-regulated the expression of dur3 and gln3,but had no effect on the expression of vacuolar membrane protein atg22,tor1,dal81 and other GATA family transcription factors.This suggests that the deletion of dal80 may promote the intracellular transport of urea but does not affect the balance of amino acids between cytoplasm and vacuoles.