Mechanism Analysis Of Nitrogen Catabolite Repression In Saccharomyces Cerevisiae And Selection Of Low Urea Producing Strain

Ethyl carbamate(EC)is a chemical compound commonly found in fermented foods and beverages.It has been verified to be a genotoxic carcinogen for human being which seriously affected the safety of our country's traditional fermented foods.Urea is the one of the most important EC precursor in yellow rice wine,therefore,decreasing the formation of urea can be an effective measure to reduce the EC content.In this study,the influences of GATA transcriptional factors,arginine transporters and Dur3 p ubiquitination on urea formation were investigated in a model haploid S.cerevisiae strain,BY4741.Furthermore,genome-wide mapping of nucleosome positions in Saccharomyces cerevisiae were analysed in response to different nitrogen conditions.On the basis of metabolic engineering,the adaptive evolution method was also used for low urea production S.cerevisiae selection.The main contents and results of this study are as follows:(1)In rich medium,the urea accumulation was positively regulated by Gln3 p and Gat1 p,and negatively regulated by Dal80 p and Gzf3 p.Gene disruption of GZF3 showed 18.7% reduction of urea accumulation.When both preferred and non-preferred nitrogen source were existed,overexpression of GLN3 and GAT1 showed little effect on urea reduction,in contrast,overexpression of DAL80 and GZF3 significantly increased the urea accumulation at the end of fermentation.Overexpression of truncated GLN3 and GAT1 could be used to reduce the urea accumulation,the urea reduction relied on the integrality of GLN3,GAT1 and DAL80 genes.The disruption of GZF3 gene further facilitated urea reduction by overexpression of truncated GLN3 and GAT1 genes.(2)ALP1 disruption showed little effect on cell growth,while the growth of CAN1 and GAP1 disruption strains was suppressed compared with wildtype(WT)strain.Overexpression of GAP1 gene showed limited effects on growth and arginine utilization,while overexpression of Alp1 p and Can1 p enhanced growth and arginine utilization in YNB medium with arginine as sole nitrogen source.Alp1 p was highly efficient for arginine utilization.However,Can1 p showed higher capacity for arginine transport.Three arginine permeases showed a synergetic effect on each other and arginine metabolism related genes.Compared to the WT strain,?gap1?can1 showed 68.4% urea reduction at the end of fermentation.(3)After submitting all of the Dur3 p residues to online prediction sofeware Ub Pred,two potential ubiquitinated lysine residues,K556,K571 were predicted.Compared with YNB + urea medium,the addition of glutamine showed increase of the relative fluorescence intensity in Dur3p(95.8%).Compared with Dur3 p,the Dur3p571 R mutant showed a 14.3% decrease of relative fluorescence intensity in YNB + urea medium.Compared with Dur3 p,the Dur3 p K556R mutant showed a 16.2% decrease of relative fluorescence intensity in YNB + urea and glutamine medium.Compared with Dur3 p,the Dur3 p K556R,Dur3 p K571R mutants showed 75.8 and 28.3% urea concentration decreases at 48 h in the ?dur3 strain in YPD medium.(4)Well-organized chromatin is involved in a number of various transcriptional regulation and gene expression.We used genome-wide mapping of nucleosomes in response to different nitrogen conditions to determine both nucleosome profiles and gene expression events in Saccharomyces cerevisiae.Nitrogen conditions influence general nucleosome profiles and the expression of nitrogen catabolite repression(NCR)sensitive genes.The nucleosome occupancy of TATA-containing genes was higher compared to TATA-less genes.TATA-less genes in high or low nucleosome occupancy,showed a significant change in gene coding regions when shifting cells from glutamine to proline as the sole nitrogen resource.Furthermore,a correlation between the expression of nucleosome occupancy induced NCR sensitive genes or TATA containing genes in NCR sensitive genes,and nucleosome prediction were found when cells were cultured in proline or shifting from glutamine to proline as the sole nitrogen source compared to glutamine.(5)Based on the adaptive evolutionary process,a mutant with low urea production was selected.Compared to the WT strain,the mutant showed 16.8% decrease of urea content in a model rice wine system.Furthermore,the genomic DNA of the mutant was sequenced using Illumina Miseq technology,and the sequencing data were used for comparative genomics analysis.Finally,170 variant were obtained,in which,27 genes with 64 amino acids change were identified.The mutated genes were resequenced and analyzed to verify the obove results.The ESL2 gene was considered as the potential genes related to urea reduction.Compared to the WT strain,disruption of ESL2 showed 12.8% decrease of urea content at the end of fermentation in YPD medium.