scholarly journals Metabolic Engineering of the Regulators in Nitrogen Catabolite Repression To Reduce the Production of Ethyl Carbamate in a Model Rice Wine System

2013 ◽  
Vol 80 (1) ◽  
pp. 392-398 ◽  
Author(s):  
Xinrui Zhao ◽  
Huijun Zou ◽  
Jianwei Fu ◽  
Jingwen Zhou ◽  
Guocheng Du ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Nuclear Localization ◽  
Catabolite Repression ◽  
Genetically Modified ◽  
Nitrogen Sources ◽  
Ethyl Carbamate ◽  
Rice Wine ◽  
Nitrogen Catabolite Repression ◽  
Wine Production ◽  
Content Type

ABSTRACTRice 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) inSaccharomyces 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 ofURE2provided an additional method of reducing urea accumulation. By combining these strategies, the genes involved in urea utilization (DUR1,2andDUR3) 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.

scholarly journals Global Expression Analysis of the Yeast Lachancea (Saccharomyces) kluyveri Reveals NewURCGenes Involved in Pyrimidine Catabolism

2013 ◽  
Vol 13 (1) ◽  
pp. 31-42 ◽  
Author(s):  
Anna Andersson Rasmussen ◽  
Dineshkumar Kandasamy ◽  
Halfdan Beck ◽  
Seth D. Crosby ◽  
Olof Björnberg ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Catabolite Repression ◽  
Nitrogen Sources ◽  
Global Changes ◽  
Nitrogen Catabolite Repression ◽  
Content Type ◽  
Double Deletion ◽  
Pyrimidine Catabolism ◽  
Saccharomyces Kluyveri ◽  
Similar Expression Pattern

ABSTRACTPyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, theURCpathway, has been initially discovered in our laboratory in the yeastLachancea kluyveri. Here, we present the global changes in gene expression inL. kluyveriin response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the knownURCgenes,URC1-6, helped to identify nine putative novelURCgenes with a similar expression pattern. The microarray analysis provided evidence that both theURCandPYDgenes are under nitrogen catabolite repression inL. kluyveriand are induced by uracil or dihydrouracil, respectively. We determined the function ofURC8, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that theL. kluyveriFui1p protein transported uridine, just like its homolog inSaccharomyces cerevisiae, but we demonstrated that is was not the only uridine transporter inL. kluyveri. We also showed that theL. kluyverihomologs ofDUR3andFUR4do not have the same function that they have inS. cerevisiae, where they transport urea and uracil, respectively. InL. kluyveri, both of these deletion strains grew normally on uracil and urea.

scholarly journals Genes of Different Catabolic Pathways Are Coordinately Regulated by Dal81 in Saccharomyces cerevisiae

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Marcos D. Palavecino ◽  
Susana R. Correa-García ◽  
Mariana Bermúdez-Moretti
Keyword(s):  
Catabolite Repression ◽  
Transcriptional Control ◽  
Nitrogen Limitation ◽  
Temporal Order ◽  
Nitrogen Sources ◽  
Nitrogen Utilization ◽  
Aminobutyric Acid ◽  
General Mechanism ◽  
Nitrogen Catabolite Repression ◽  
Catabolic Enzymes

Yeast can use a wide variety of nitrogen compounds. However, the ability to synthesize enzymes and permeases for catabolism of poor nitrogen sources is limited in the presence of a rich one. This general mechanism of transcriptional control is called nitrogen catabolite repression. Poor nitrogen sources, such as leucine, γ-aminobutyric acid (GABA), and allantoin, enable growth after the synthesis of pathway-specific catabolic enzymes and permeases. This synthesis occurs only under conditions of nitrogen limitation and in the presence of a pathway-specific signal. In this work we studied the temporal order in the induction of AGP1, BAP2, UGA4, and DAL7, genes that are involved in the catabolism and use of leucine, GABA, and allantoin, three poor nitrogen sources. We found that when these amino acids are available, cells will express AGP1 and BAP2 in the first place, then DAL7, and at last UGA4. Dal81, a general positive regulator of genes involved in nitrogen utilization related to the metabolisms of GABA, leucine, and allantoin, plays a central role in this coordinated regulation.

scholarly journals The Yeast GATA Factor Gat1 Occupies a Central Position in Nitrogen Catabolite Repression-Sensitive Gene Activation

2009 ◽  
Vol 29 (13) ◽  
pp. 3803-3815 ◽  
Author(s):  
Isabelle Georis ◽  
André Feller ◽  
Fabienne Vierendeels ◽  
Evelyne Dubois
Keyword(s):  
Gene Expression ◽  
Catabolite Repression ◽  
Nitrogen Availability ◽  
Gene Activation ◽  
Nitrogen Sources ◽  
Central Position ◽  
Limiting Factor ◽  
Nitrogen Catabolite Repression ◽  
Gata Factor ◽  
Gata Factors

ABSTRACT Saccharomyces cerevisiae cells are able to adapt their metabolism according to the quality of the nitrogen sources available in the environment. Nitrogen catabolite repression (NCR) restrains the yeast's capacity to use poor nitrogen sources when rich ones are available. NCR-sensitive expression is modulated by the synchronized action of four DNA-binding GATA factors. Although the first identified GATA factor, Gln3, was considered the major activator of NCR-sensitive gene expression, our work positions Gat1 as a key factor for the integrated control of NCR in yeast for the following reasons: (i) Gat1 appeared to be the limiting factor for NCR gene expression, (ii) GAT1 expression was regulated by the four GATA factors in response to nitrogen availability, (iii) the two negative GATA factors Dal80 and Gzf3 interfered with Gat1 binding to DNA, and (iv) Gln3 binding to some NCR promoters required Gat1. Our study also provides mechanistic insights into the mode of action of the two negative GATA factors. Gzf3 interfered with Gat1 by nuclear sequestration and by competition at its own promoter. Dal80-dependent repression of NCR-sensitive gene expression occurred at three possible levels: Dal80 represses GAT1 expression, it competes with Gat1 for binding, and it directly represses NCR gene transcription.

scholarly journals Gat1p, a GATA family protein whose production is sensitive to nitrogen catabolite repression, participates in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (3) ◽  
pp. 847-858 ◽  
Author(s):  
J A Coffman ◽  
R Rai ◽  
T Cunningham ◽  
V Svetlov ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Catabolite Repression ◽  
Nitrogen Sources ◽  
Nitrogen Catabolite Repression ◽  
Triple Mutant ◽  
Regulatory Circuit ◽  
Catabolic Genes ◽  
Family Protein ◽  
Sequence Elements

Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.

scholarly journals A Bacillus paralicheniformis Iron-Containing Urease Reduces Urea Concentrations in Rice Wine

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Qingtao Liu ◽  
Yuqi Chen ◽  
Minglai Yuan ◽  
Guocheng Du ◽  
Jian Chen ◽  
...  
Keyword(s):  
Food Industry ◽  
Ethyl Carbamate ◽  
Rice Wine ◽  
Enzymatic Assays ◽  
Content Type ◽  
Urease Gene ◽  
Bacillus Paralicheniformis ◽  
Acid Urease ◽  
History Of ◽  
Insight Into

ABSTRACT Urease, a nickel-containing metalloenzyme, was the first enzyme to be crystallized and has a prominent position in the history of biochemistry. In the present study, we identified a nickel urease gene cluster, ureABCEFGDH, in Bacillus paralicheniformis ATCC 9945a and characterized it in Escherichia coli. Enzymatic assays demonstrate that this oxygen-stable urease is also an iron-containing acid urease. Heterologous expression assays of UreH suggest that this accessory protein is involved in the transmembrane transportation of nickel and iron ions. Moreover, this iron-containing acid urease has a potential application in the degradation of urea in rice wine. The present study not only enhances our understanding of the mechanism of activation of urease but also provides insight into the evolution of metalloenzymes. IMPORTANCE An iron-containing, oxygen-stable acid urease from B. paralicheniformis ATCC 9945a with good enzymatic properties was characterized. This acid urease shows activities toward both urea and ethyl carbamate. After digestion with 6 U/ml urease, approximately 92% of the urea in rice wine was removed, suggesting that this urease has great potential in the food industry.

scholarly journals Multiple Nuclear Localization Signals Mediate Nuclear Localization of the GATA Transcription Factor AreA

2014 ◽  
Vol 13 (4) ◽  
pp. 527-538 ◽  
Author(s):  
Cameron C. Hunter ◽  
Kendra S. Siebert ◽  
Damien J. Downes ◽  
Koon Ho Wong ◽  
Sara D. Kreutzberger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Nitrogen Sources ◽  
Nuclear Accumulation ◽  
Content Type ◽  
Nuclear Localization Signals ◽  
Gata Transcription Factor

ABSTRACTTheAspergillus nidulansGATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in theSaccharomyces cerevisiaefunctional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructedareAmutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors fromAspergillus, yeast, and mammals, indicating an ancient origin.

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