Identification Of Key Transcription Factors In Saccharomyces Cerevisiae Nitrogen Metabolism And Investigation Of Their Regulatory Mechanisms

The nitrogen metabolism regulatory network of Saccharomyces cerevisiae has received extensive attention.The in-depth and systematic study of the complex nitrogen metabolism regulatory network can help metabolize the engineered S.cerevisiae strains and serve as a reference model for understanding the nitrogen metabolism regulatory network of other microorganisms.In this study,we experimentally investigated the utilization of 30 nitrogen sources by S.cerevisiae S288 c and gene expression changes under different nitrogen source conditions.On this basis,by constructing a gene co-expression network,we verified that the chromatin regulator Eaf3 p,endocytosis and mitochondrial-related hub genes have regulatory effects on the nitrogen metabolism of S.cerevisiae.Further studies screened and validated different chromatin regulators in response to different nitrogen sources,and identified Ahc1 p,a chromatin regulator with a global regulatory effect on S.cerevisiae nitrogen metabolism.Finally,the mechanisms of Eaf3 p and Ahc1 p regulating nitrogen metabolism were deeply studied.This study explored the global regulators of nitrogen metabolism and their regulatory mechanisms in S.cerevisiae,which is of great significance for revealing the regulatory network of nitrogen metabolism in S.cerevisiae and the metabolic transformation of industrial S.cerevisiae.The main results of this paper are as follows:1.Identification of the regulatory effect of chromatin regulator Eaf3 p and other hub genes on S.cerevisiae S288 c nitrogen metabolism.To screen the regulators that play key roles in S.cerevisiae nitrogen metabolism,30 different nitrogen sources were used for short-term impact on S.cerevisiae S288 c,and then transcriptome analysis was conducted.Then,a gene coexpression network was constructed based on the transcriptome data,and 10 hub genes located at the center of the network were identified.The functional verification of the hub genes revealed that the chromatin regulator Eaf3 p coding gene,endocytosis-related gene END3,mitochondrial-related genes CAP1,ATP12,NAM9,MRPL22 and MRP1 play essential roles in regulating nitrogen metabolism in S.cerevisiae.Overexpression of these genes reduced urea accumulation of S288 c without significantly affecting its growth,and significantly increased the utilization of a variety of non-preferred nitrogen sources by co-regulating amino acid transporters,nitrogen catabolism repression(NCR)activating factors and other key nitrogen metabolism pathways of S288 c.2.Identification and verification of the global regulatory effect of chromatin regulator Ahc1 p on S.cerevisiae S288 c nitrogen metabolism.After discovering that the chromatin regulator Eaf3 p plays an essential role in regulating S.cerevisiae nitrogen metabolism.To further explore the chromatin regulators that regulate nitrogen metabolism,the transcriptome data of S.cerevisiae S288 c under different nitrogen conditions were compared.The results showed that the expression difference between chromatin regulators and NCR genes was consistent under different nitrogen sources.The functional analysis identified that chromatin regulator Ahc1 p global regulates S.cerevisiae nitrogen metabolism.The results revealed that Ahc1 p significantly increased the utilization rate of various amino acids and reduced the accumulation of harmful nitrogen metabolite precursor urea without affecting the growth of the strain by simultaneously regulating the Ssy1p-Ptr3p-Ssy5 p sensing system,amino acid transporters,NCR activators,and general amino acid control pathway.3.The key regulator,Eaf3 p,acts as a trans-acting factor to regulate S.cerevisiae S288 c nitrogen metabolism.After finding that Eaf3 p is a crucial regulator of nitrogen metabolism in S.cerevisiae,the mechanism of nitrogen metabolism regulation was deeply studied and analyzed.By investigating the effect of overexpressing EAF3 on histone H4 acetylation levels at its target gene promoters,we found that Eaf3 p may regulate the expression of target genes by regulating the histone acetylation level of its target gene promoter through the function of its histone acetyltransferase Nu A4 complex.Further experimental studies were carried out on the binding sites of Eaf3 p on its target gene promoters and its interacting transcription factors.It was determined that Eaf3 p directly regulates nitrogen metabolism relate genes GAT1,CAN1,LYP1,GLN3 and HIP1 through its binding on their promoters.This study also revealed that Eaf3 p was recruited with Pho2 p or other transcription factors on the enhancer to promote the expression of target genes.4.The global regulator Ahc1 p acts as a trans-acting factor or transcription cofactor to regulate nitrogen metabolism in S.cerevisiae S288 c.Based on the above findings that the chromatin regulator Ahc1 p is involved in the global regulation of nitrogen metabolism in S.cerevisiae,this section explored the mechanism of its regulation of nitrogen metabolism.By studying the binding of Ahc1 p to the promoters of nitrogen metabolism-related genes it regulates,it was determined that Ahc1 p directly regulates several nitrogen metabolism genes by binding to their promoters.Further study on the binding site of Ahc1 p on the target gene promoters and the transcription factors interacting with Ahc1 p revealed that Ahc1 p might regulate some nitrogen metabolism genes in two ways.First,as a cofactor,Ahc1 p combines with transcription factors such as Rtg3 p or Gcr1 p to promote the binding of the transcription complex to the core promoters of nitrogen metabolism-related genes TAT1,GCN4 and GLN3,and promote their transcription initiation.Second,Ahc1 p and transcription factors recruit each other on enhancers to promote the transcription of nitrogen metabolism-related genes GAP1,MUP1,STP1,SSY5 and DUR1,2.