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Zhang P.,Jiangnan University | Du G.,Jiangnan University | Zou H.,Zhejiang Guyuelongshan Shaoxing Wine Company | Xie G.,Zhejiang Guyuelongshan Shaoxing Wine Company | And 3 more authors.
Journal of Agricultural and Food Chemistry | Year: 2017

Ubiquitination can significantly affect the endocytosis and degradation of plasma membrane proteins. Here, the ubiquitination of a Saccharomyces cerevisiae urea plasma membrane transporter (Dur3p) was altered. Two potential ubiquitination sites, lysine residues K556 and K571, of Dur3p were predicted and replaced by arginine, and the effects of these mutations on urea utilization and formation under different nitrogen conditions were investigated. Compared with Dur3p, the Dur3pK556R mutant showed a 20.1% decrease in ubiquitination level in yeast nitrogen base medium containing urea and glutamine. It also exhibited a >75.8% decrease in urea formation in yeast extract-peptone-dextrose medium and 41.3 and 55.4% decreases in urea and ethyl carbamate formation (a known carcinogen), respectively, in a model rice wine system. The results presented here show that the mutation of Dur3p ubiquitination sites could significantly affect urea utilization and formation. Modifying the ubiquitination of specific transporters might have promising applications in rationally engineering S. cerevisiae strains to efficiently use specific nitrogen sources. © 2017 American Chemical Society.


Cao Y.,Jiangnan University | Xie G.,Zhejiang Guyue Longshan Shaoxing Wine Co. | Xie G.,Tech Lab Group | Wu C.,Jiangnan University | Lu J.,Jiangnan University
Journal of the Institute of Brewing | Year: 2010

The objective of the study was to systematically investigate flavor compounds in Chinese rice wine (CRW) using chromatography technology. In twelve CRW samples, 93 different flavor compounds were detected and identified including 16 alcohols in addition to ethanol, 29 esters, 9 aldehydes, 9 organic acids, 19 amino acids and 11 fatty acids. Statistical analysis by principal component analysis (PCA) indicated that seventeen flavor compounds in Guyue Longshan rice wine made a large contribution to its special flavor. These compounds were benzaldehyde, acetaldehyde, ethyl 2-hydroxy-4-methylvalerate, ethyl butyrate, phenyl ethyl isobutyrate, ethyl benzoate, ethyl phenylacetate, methyl dodecanoate, methyl oleate, ethyl dedecanoate, 1-butanol, 3-ethoxyl-1-propanol, 1-enanthol, dodecanol, lactic acid, fumaric acid and lauric acid. © 2010 The Institute of Brewing & Distilling.


Zhao S.,Jiangnan University | Zhao X.,Jiangnan University | Zou H.,Zhejiang Guyuelongshan Shaoxing Wine Company | Fu J.,Zhejiang Guyuelongshan Shaoxing Wine Company | And 3 more authors.
Journal of Proteomics | Year: 2014

In cultures containing multiple sources of nitrogen, Saccharomyces cerevisiae exhibits a sequential use of nitrogen sources through a mechanism known as nitrogen catabolite repression (NCR). To identify proteins differentially expressed due to NCR, proteomic analysis of S. cerevisiae S288C under different nitrogen source conditions was performed using two-dimensional gel electrophoresis (2-DE), revealing 169 candidate protein spots. Among these 169 protein spots, 121 were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF). The identified proteins were closely associated with four main biological processes through Gene Ontology (GO) categorical analysis. The identification of the potential proteins and cellular processes related to NCR offer a global overview of changes elicited by different nitrogen sources, providing clues into how yeast adapt to different nutritional conditions. Moreover, by comparing our proteomic data with corresponding mRNA data, proteins regulated at the transcriptional and post-transcriptional level could be distinguished. Biological significance. In S. cerevisiae, different nitrogen sources provide different growth characteristics and generate different metabolites. The nitrogen catabolite repression (NCR) process plays an important role for S. cerevisiae in the ordinal utilization of different nitrogen sources. NCR process can result in significant shift of global metabolic networks. Previous works on NCR primarily focused on transcriptomic level. The results obtained in this study provided a global atlas of the proteome changes triggered by different nitrogen sources and would facilitate the understanding of mechanisms for how yeast could adapt to different nutritional conditions. © 2014 Elsevier B.V.


Zhao X.,Jiangnan University | Zou H.,Zhejiang Guyuelongshan Shaoxing Wine Company | Fu J.,Zhejiang Guyuelongshan Shaoxing Wine Company | Zhou J.,Jiangnan University | And 2 more authors.
Applied and Environmental Microbiology | Year: 2014

Rice wine has been one of the most popular traditional alcoholic drinks in China. However, the presence of potentially carcinogenic ethyl carbamate (EC) in rice wine has raised a series of food safety issues. During rice wine production, the key reason for EC formation is urea accumulation, which occurs because of nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. NCR represses urea utilization by retaining Gln3p in the cytoplasm when preferred nitrogen sources are present. In order to increase the nuclear localization of Gln3p, some possible phosphorylation sites on the nuclear localization signal were mutated and the nuclear localization regulation signal was truncated, and the disruption of URE2 provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2 and DUR3) could be significantly activated in the presence of glutamine. During shake flask fermentations of the genetically modified strains, very little urea accumulated in the medium. Furthermore, the concentrations of urea and EC were reduced by 63% and 72%, respectively, in a model rice wine system. Examination of the normal nutrients in rice wine indicated that there were few differences in fermentation characteristics between the wild-type strain and the genetically modified strain. These results show that metabolic engineering of the NCR regulators has great potential as a method for eliminating EC during rice wine production. © 2014, American Society for Microbiology.


Zhao X.,Jiangnan University | Zou H.,Zhejiang Guyuelongshan Shaoxing Wine Company | Fu J.,Zhejiang Guyuelongshan Shaoxing Wine Company | Chen J.,Jiangnan University | And 2 more authors.
Yeast | Year: 2013

Rice wine is a popular traditional alcoholic drink with a long history in China. However, the presence of the potential carcinogen ethyl carbamate (EC) raises a series of food safety concerns. Although the metabolic pathway of urea (the major precusor of EC) has been characterized in Saccharomyces cerevisiae, the regulation of urea accumulation remains unclear, making the efficient elimination of urea difficult. To demonstrate the regulatory mechanisms governing urea accumulation, three key nitrogen sources that can inhibit urea utilization for a commercial S. cerevisiae strain were identified. In addition, regulators of nitrogen catabolite repression (NCR) and target of rapamycin (TOR) pathways were identified as being involved in urea accumulation by real-time quantitative PCR. Based on these results, preferred nitrogen sources were found to repress urea utilization by converting them to glutamine or glutamate. Moreover, the results indicated that the manner of urea metabolism regulation was different for two positive regulators involved in NCR; Gln3p can be retained in the cytoplasm by glutamine, while Gat1p can be retained by glutamine and glutamate. Furthermore, this was confirmed by fluorescence location detection. These new findings provide new targets for eliminating EC and other harmful nitrogen-containing compounds in fermented foods. © 2013 John Wiley & Sons, Ltd.


Zhao X.,Jiangnan University | Du G.,Jiangnan University | Zou H.,Zhejiang Guyuelongshan Shaoxing Wine Company | Fu J.,Zhejiang Guyuelongshan Shaoxing Wine Company | And 2 more authors.
Trends in Food Science and Technology | Year: 2013

Ethyl carbamate (EC) is a potentially carcinogenic compound that is widely found in alcoholic beverages. Because of its toxicity, carcinogenicity, and universality, EC is currently one of the biggest challenges in the alcoholic beverages industry. Many methods for reducing the EC level have been investigated, incorporating physical, chemical, enzymatic, and metabolic engineering technologies. This review focuses on the traditional methods as well as several new methods. Furthermore, by comparing the advantages and disadvantages of these methods, various issues are addressed relating to the development of measures to eliminate EC in alcoholic beverages on a laboratory scale and an industrial scale. © 2013 Elsevier Ltd.


PubMed | Zhejiang Guyuelongshan Shaoxing Wine Company and Jiangnan University
Type: | Journal: Scientific reports | Year: 2016

In Saccharomyces cerevisiae, when preferred nitrogen sources are present, the metabolism of non-preferred nitrogen is repressed. Previous work showed that this metabolic regulation is primarily controlled by nitrogen catabolite repression (NCR) related regulators. Among these regulators, two positive regulators (Gln3p and Gat1p) could be phosphorylated and sequestered in the cytoplasm leading to the transcription of non-preferred nitrogen metabolic genes being repressed. The nuclear localization signals (NLSs) and nuclear localization regulatory signals (NLRSs) in Gln3p and Gat1p play essential roles in the regulation of their localization in cells. However, compared with Gln3p, the information of NLS and NLRS for Gat1p remains unknown. In this study, residues 348-375 and 366-510 were identified as the NLS and NLRS of Gat1p firstly. In addition, the modifications of Gat1p (mutations on the NLS and truncation on the NLRS) were attempted to enhance the transcription of non-preferred nitrogen metabolic genes. Quantitative real-time PCR showed that the transcriptional levels of 15 non-preferred nitrogen metabolic genes increased. Furthermore, during the shaking-flask culture tests, the utilization of urea, proline and allantoine was significantly increased. Based on these results, the genetic engineering on Gat1p has a great potential in enhancing non-preferred nitrogen metabolism in S. cerevisiae.


PubMed | Zhejiang Guyuelongshan Shaoxing Wine Company and Jiangnan University
Type: | Journal: Scientific reports | Year: 2016

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. These results also showed that variation of nucleosome occupancy accompany with chromatin-dependent transcription factor could influence the expression of a series of genes involved in the specific regulation of nitrogen utilization.


PubMed | Zhejiang Guyuelongshan Shaoxing Wine Company and Jiangnan University
Type: | Journal: Journal of proteomics | Year: 2014

In cultures containing multiple sources of nitrogen, Saccharomyces cerevisiae exhibits a sequential use of nitrogen sources through a mechanism known as nitrogen catabolite repression (NCR). To identify proteins differentially expressed due to NCR, proteomic analysis of S. cerevisiae S288C under different nitrogen source conditions was performed using two-dimensional gel electrophoresis (2-DE), revealing 169 candidate protein spots. Among these 169 protein spots, 121 were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF). The identified proteins were closely associated with four main biological processes through Gene Ontology (GO) categorical analysis. The identification of the potential proteins and cellular processes related to NCR offer a global overview of changes elicited by different nitrogen sources, providing clues into how yeast adapt to different nutritional conditions. Moreover, by comparing our proteomic data with corresponding mRNA data, proteins regulated at the transcriptional and post-transcriptional level could be distinguished. Biological significance In S. cerevisiae, different nitrogen sources provide different growth characteristics and generate different metabolites. The nitrogen catabolite repression (NCR) process plays an important role for S. cerevisiae in the ordinal utilization of different nitrogen sources. NCR process can result in significant shift of global metabolic networks. Previous works on NCR primarily focused on transcriptomic level. The results obtained in this study provided a global atlas of the proteome changes triggered by different nitrogen sources and would facilitate the understanding of mechanisms for how yeast could adapt to different nutritional conditions.


PubMed | Zhejiang Guyuelongshan Shaoxing Wine Company and Jiangnan University
Type: | Journal: Scientific reports | Year: 2016

Arginine plays an important role in cellular function and metabolism. Arginine uptake mainly occurs through three amino acid permeases, Alp1p, Gap1p and Can1p, which act as both transporters and receptors for amino acid utilization. In this study, seven mutants were constructed with different combinations of permease deficiencies that inhibit arginine utilization. Their effects on arginine metabolism were measured. The three amino acid permeases were also individually overexpressed in wild-type (WT), alp1gap1can1 and npr1 strains. The growth and arginine utilization of can1, gap1can1 and alp1gap1can1 mutants were suppressed in YNB medium when arginine was the sole nitrogen source. Meanwhile, overexpression of Alp1p and Can1p enhanced growth and arginine utilization in WT, alp1gap1can1 and npr1. Besides, overexpression of Can1p caused a 26.7% increase in OD600 and 29.3% increase in arginine utilization compared to that of Alp1p in alp1gap1can1. Transcription analysis showed that the effects of three amino acid permeases on the arginine utilization and the regulation of related genes, were tightly related to their individual characteristics. However, their overall effects were different for different combinations of mutants. The results presented here suggest some possible synergistic effects of different amino acid permeases on regulation of amino acid utilization and metabolism.

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