Filamentation of Metabolic Enzymes in Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Shen QJ
PROVIDER: S-EPMC4920916 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Asparagine synthetase (ASNS) and CTP synthase (CTPS) are two metabolic enzymes crucial for glutamine homeostasis. A genome-wide screening in Saccharomyces cerevisiae reveal that both ASNS and CTPS form filamentous structures termed cytoophidia. Although CTPS cytoophidia were well documented in recent years, the filamentation of ASNS is less studied. Using the budding yeast as a model system, here we confirm that two ASNS proteins, Asn1 and Asn2, are capable of forming cytoophidia in diauxic and stationary phases. We find that glucose deprivation induces ASNS filament formation. Although ASNS and CTPS form distinct cytoophidia with different lengths, both structures locate adjacently to each other in most cells. Moreover, we demonstrate that the Asn1 cytoophidia colocalize with the Asn2 cytoophidia, while Asn2 filament assembly is largely dependent on Asn1. In addition, we are able to alter Asn1 filamentation by mutagenizing key sites on the dimer interface. Finally, we show that ASN1D330V promotes filamentation. The ASN1D330V mutation impedes cell growth in an ASN2 knockout background, while growing normally in an ASN2 wild-type background. Together, this study reveals a connection between ASNS and CTPS cytoophidia and the differential filament-forming capability between two ASNS paralogs.
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Project description:The Saccharomyces cerevisiae transcription factor Ste12 controls two distinct developmental programs of mating and filamentation. Ste12 activity is regulated by Fus3 and Kss1 mitogen-activated protein kinases through two Ste12 inhibitors, Dig1 and Dig2. Mating genes are regulated by Ste12 through Ste12 binding sites (pheromone response elements [PREs]), whereas filamentation genes are supposedly regulated by the cooperative binding of Ste12 and Tec1 on a PRE adjacent to a Tec1-binding site (TCS), termed filamentous responsive element (FRE). However, most filamentation genes do not contain an FRE; instead, they all have a TCS. By immunoprecipitation, we show that Ste12 forms two distinct complexes, Ste12/Dig1/Dig2 and Tec1/Ste12/Dig1, both in vivo and in vitro. The two complexes are formed by the competitive binding of Tec1 and Dig2 with Ste12, as Tec1 can compete off Dig2 from Ste12 in vitro and in vivo. In the Tec1/Ste12/Dig1 complex, Tec1 binds to the N terminus of Ste12 and to Dig1 indirectly through Ste12. Tec1 has low basal activity, and its transcriptional activation is provided by the associated Ste12, which is under Dig1 inhibition. Filamentation genes are bound by the Tec1/Ste12/Dig1 complex, whereas mating genes are occupied by mostly Ste12/Dig1/Dig2 with some Tec1/Ste12/Dig1. We suggest that Tec1 tethers Ste12 to TCS elements upstream of filamentation genes and defines the filamentation genes as a subset of Ste12-regulated genes.
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Project description:Iron is an essential cofactor for enzymes involved in numerous cellular processes, yet little is known about the impact of iron deficiency on cellular metabolism or iron proteins. Previous studies have focused on changes in transcript and proteins levels in iron-deficient cells, yet these changes may not reflect changes in transport activity or flux through a metabolic pathway. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Iron deficiency led to changes in glucose metabolism, amino acid biosynthesis, and lipid biosynthesis that were due to deficiencies in specific iron-dependent enzymes. Iron-sulfur proteins exhibited loss of iron cofactors, yet amino acid synthesis was maintained. Ergosterol and sphingolipid biosynthetic pathways had blocks at points where heme and diiron enzymes function, whereas Ole1, the essential fatty acid desaturase, was resistant to iron depletion. Iron-deficient cells exhibited depletion of most iron enzyme activities, but loss of activity during iron deficiency did not consistently disrupt metabolism. Amino acid homeostasis was robust, but iron deficiency impaired lipid synthesis, altering the properties and functions of cellular membranes.
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Project description:Yeast cells respond to heat stress by remodeling their gene expression, resulting in the changes of the corresponding proteins and metabolites. Compared to the intensively investigated transcriptome and proteome, the metabolic response to heat stress is not sufficiently characterized. Mitochondria have been recognized to play an essential role in heat stress tolerance. Given the compartmentalization of the cell, it is not clear if the heat stress-induced metabolic response occurs in mitochondria or in the cytosol. Therefore, a compartment-specific metabolite analysis was performed to analyze the heat stress-induced metabolic response in mitochondria and the cytoplasm. In this work, the isolated mitochondria and the cytoplasm of yeast cells grown at permissive temperature and cells adapting to heat stress were subjected to mass spectrometry-based metabolomics. Over a hundred metabolites could be identified, covering amino acid metabolism, energy metabolism, arginine metabolism, purine and pyrimidine metabolism, and others. Highly accumulated citrulline and reduced arginine suggested remodeled arginine metabolism. A stable isotope-labeled experiment was performed to analyze the heat stress-induced metabolic remodeling of the arginine metabolism, identifying activated de novo ornithine biosynthesis to support arginine and spermidine synthesis. The short-term increased spermidine and trehalose suggest their important roles as heat stress markers. These data provide metabolic clues of heat stress-induced metabolic remodeling, which helps in understanding the heat stress response.
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Project description:The roles of duplicate genes and their contribution to the phenomenon of enzyme dispensability are a central issue in molecular and genome evolution. A comprehensive classification of the mechanisms that may have led to their preservation, however, is currently lacking. In a systems biology approach, we classify here back-up, regulatory, and gene dosage functions for the 105 duplicate gene families of Saccharomyces cerevisiae metabolism. The key tool was the reconciled genome-scale metabolic model iLL672, which was based on the older iFF708. Computational predictions of all metabolic gene knockouts were validated with the experimentally determined phenotypes of the entire singleton yeast library of 4658 mutants under five environmental conditions. iLL672 correctly identified 96%-98% and 73%-80% of the viable and lethal singleton phenotypes, respectively. Functional roles for each duplicate family were identified by integrating the iLL672-predicted in silico duplicate knockout phenotypes, genome-scale carbon-flux distributions, singleton mutant phenotypes, and network topology analysis. The results provide no evidence for a particular dominant function that maintains duplicate genes in the genome. In particular, the back-up function is not favored by evolutionary selection because duplicates do not occur more frequently in essential reactions than singleton genes. Instead of a prevailing role, multigene-encoded enzymes cover different functions. Thus, at least for metabolism, persistence of the paralog fraction in the genome can be better explained with an array of different, often overlapping functional roles.
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Project description:Currently, pursuing yeast strains that display both a high potential fitness for alcoholic fermentation and a favorable impact on quality is a major goal in the alcoholic beverage industry. This considerable industrial interest has led to many studies characterizing the phenotypic and metabolic traits of commercial yeast populations. In this study, 20 Saccharomyces cerevisiae strains from different geographical origins exhibited high phenotypic diversity when their response to nine biotechnologically relevant conditions was examined. Next, the fermentation fitness and metabolic traits of eight selected strains with a unique phenotypic profile were evaluated in a high-sugar synthetic medium under two nitrogen regimes. Although the strains exhibited significant differences in nitrogen requirements and utilization rates, a direct relationship between nitrogen consumption, specific growth rate, cell biomass, cell viability, acetic acid and glycerol formation was only observed under high-nitrogen conditions. In contrast, the strains produced more succinic acid under the low-nitrogen regime, and a direct relationship with the final cell biomass was established. Glucose and fructose utilization patterns depended on both yeast strain and nitrogen availability. For low-nitrogen fermentation, three strains did not fully degrade the fructose. This study validates phenotypic and metabolic diversity among commercial wine yeasts and contributes new findings on the relationship between nitrogen availability, yeast cell growth and sugar utilization. We suggest that measuring nitrogen during the stationary growth phase is important because yeast cells fermentative activity is not exclusively related to population size, as previously assumed, but it is also related to the quantity of nitrogen consumed during this growth phase.
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Project description:Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.
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Project description:A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 µ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering.
| S-EPMC3070743 | biostudies-literature
Project description:IntroductionThe switch from quiescence (G0) into G1 and cell cycle progression critically depends on specific nutrients and metabolic capabilities. Conversely, metabolic networks are regulated by enzyme-metabolite interaction and transcriptional regulation that lead to flux modifications to support cell growth. How cells process and integrate environmental information into coordinated responses is challenging to analyse and not yet described quantitatively.ObjectivesTo quantitatively monitor the central carbon metabolism during G0 exit and the first 2 h after reentering the cell cycle from synchronized Saccharomyces cerevisiae.MethodsDynamic tailored 13C metabolic flux analysis was used to observe the intracellular metabolite flux changes, and the metabolome and proteome were observed to identify regulatory mechanisms.ResultsG0 cells responded immediately to an extracellular increase of glucose. The intracellular metabolic flux changed in time and specific events were observed. High fluxes into trehalose and glycogen synthesis were observed during the G0 exit. Both fluxes then decreased, reaching a minimum at t?=?65 min. Here, storage degradation contributed significantly (i.e. 21%) to the glycolytic flux. In contrast to these changes, the glucose uptake rate remained constant after the G0 exit. The flux into the oxidative pentose phosphate pathway was highest (29-fold increase, 36.4% of the glucose uptake) at t?=?65 min, while it was very low at other time points. The maximum flux seems to correlate with a late G1 state preparing for the S phase transition. In the G1/S phase (t?=?87 min), anaplerotic reactions such as glyoxylate shunt increased. Protein results show that during this transition, proteins belonging to clusters related with ribosome biogenesis and assembly, and initiation transcription factors clusters were continuously synthetised.ConclusionThe intracellular flux distribution changes dynamically and these major rearrangements highlight the coordinate reorganization of metabolic flux to meet requirements for growth during different cell state.
| S-EPMC6715666 | biostudies-literature
Project description:Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g., tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.
| S-EPMC4136831 | biostudies-literature