Inhibitory effects of galacturonic acid on Saccharomyces cerevisiae: involvement of hexose transporters
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Jean-Marc Daran
PROVIDER: E-GEOD-43474 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The multi-component global regulator Velvet complex has been identified as a key regulator of secondary metabolite production in Aspergillus and Penicillium species. Previous work indicated a massive impact of PcvelA and PclaeA deletions, two key components of the Velvet complex, on penicillin production in prolonged batch cultures of P. chrysogenum, as well as substantial changes in transcriptome. The present study investigates the impact of these mutations on product formation and genome-wide transcript profiles under glucose-limited, aerobic conditions, relevant for industrial production of ?-lactams. The gene-deletion cassette for PcvelA or PclaeA was integrated in a hdfA mutant of the penicillin high-producing strain P. chrysogenum DS17690. Predicted amino acid sequences of PcVelA and PcLaeA in this strain were identical to those in its ancestor Wisconsin54-1255. Controls were performed to rule out transformation-associated loss of penicillin-biosynthesis clusters which, in preliminary studies, led to a massive reduction of penicillin production in a PcvelA deletion mutant. The correct PcvelA and PclaeA deletion strains revealed a significant (up to 30 %) reduction of penicillin-G productivity relative to the reference strain, which is a much smaller reduction than previously reported for prolonged batch cultures of P. chrysogenum strains. Chemostat-based transcriptome analysis yielded only 23 genes with a consistent response in the PcvelA? and PclaeA? mutants when grown in the absence of the penicillin-G side-chain precursor phenylacetic acid. 11 of these genes belonged to two small gene clusters (with 5 and 6 genes, respectively), one of which contains a gene with high homology to an aristolochene synthase. These results provide a clear caveat that the impact of the Velvet complex on secondary metabolism in filamentous fungi may be strongly context dependent Previous studies on the impact of the Velvet complex in P. chrysogenum were performed in prolonged batch cultures. Time course analysis revealed that the impact of PcvelA and PclaeA mutations was most pronounced after prolonged incubation {Hoff, 2010 6 /id}, but the physiological status of these cultures was not precisely defined. Industrial production of ?-lactam antibiotics is performed in sugar-limited, aerobic fed-batch cultures {Menezes, 1994 76 /id}. The aim of the present study is to investigate the impact of the Velvet complex on physiology, penicilllin production and transcriptional regulation under industrially relevant conditions. To this end, we studied the impact of PcvelA and PclaeA deletions in aerobic, glucose-limited chemostat cultures of the penicillin high-producing strain P. chrysogenum DS17690.
2011-12-06 | E-GEOD-31633 | biostudies-arrayexpress
Project description:Industrial production of penicillin G by Penicillium chrysogenum requires medium supplementation with the side chain precursor phenylacetate. However, P.chrysogenum grown in presence of phenylalanine as sole nitrogen source formed detectable extracellular amounts of phenylacetate and penicillin G. To get more insights in the metabolism implicated, chemostat-cultivation in presence of 13C9-phenylalanine were carried out. Quantification and modeling of the labeled metabolite pools indicated that phenylalanine was i) incorporated in nascent protein, ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation and iii) oxidized into tyrosine and subsequently assimilated in the homogentisate pathway. Comparative transcriptome analysis of phenylalanine and (NH4)2SO4 grown P.chrysogenum cultures enabled to identify two putative 2-oxo acid decarboxylases Pc13g9300 and Pc18g01490. Both cDNAs were cloned and expressed in the decarboxylase-free Saccharomyces cerevisiae CEN.PK711-7C (pdc1delta, 5delta, 6delta, aro10delta, thi3delta) strain that has lost the ability to grow on glucose as sole carbon source or on phenylalanine as sole nitrogen source. Only Pc13g09300 was able to restore growth on glucose and on phenylalanine, demonstrating that this gene encodes a dual substrate pyruvate, phenylpyruvate decarboxylase. This newly identified thiamine-dependent 2-oxo acid decarboxylase provides clues to explain the formation of phenylacetate via an Ehrlich-like pathway in P.chrysogenum. Penicillium chrysogenum DS17690 was grown in aerobic glucose limited chemostat at 25oC, pH 6.5 and a dilution rate of 0.03h-1 with either amonia or phenylalanine
2011-11-05 | E-GEOD-32097 | biostudies-arrayexpress
Project description:Background: Microbial gene expression is to a large extend determined by environmental growth conditions. Differential gene expression analysis between two conditions has been frequently used to reveal regulatory networks and to assign physiological function to unknown genes. In nature, microorganisms cohabit however these interactions have been rarely studied and reproduced in laboratory set-up. Thus to quantitatively explore the genome-wide responses of microbial interaction, we co-cultivated Penicillium chrysogenum and Bacillus subtilis in chemostat culture. Results: Time course expression analysis of P. chrysogenum to co-cultivation with B. subtilis was carried out to understand the natural responses of P. chrysogenum to prokaryotes. Steady state chemostats of P. chrysogenum in non-B-lactam producing conditions was pulsed with B. subtilis and co-cultivation was followed for 72 hours. The dynamic physiological and transcriptional responses of P. chrysogenum in mixed culture were monitored. B. subtilis outcompeted growth of P. chrysogenum resulting in an increased B. subtilis biomass by more than three fold of its original size and a reduction in P. chrysogenum biomass to half of its original size after 72 h of mixed culture. Genes of the penicillin pathway, synthesis of the side-chain and precursors were overall unresponsive to the presence of B. subtilis. Moreover Penicillium polyketide synthase and nonribosomal peptide synthetase genes either remained silent or down-regulated, whereas genes responsible for protein synthesis, metabolism, energy conservation, respiration and transport were upregulated in the presence of B. subtilis. Among highly responsive genes, two putative B-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production in P. chrysogenum. Mutanase activity was not observed in pure cultures of P. chrysogenum and B. subtilis or when P. chrysogenum was co-cultured with B. subtilis supernatant or heat inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum pulsed with filter sterilized supernatants from mixed cultures P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that at least Pc12g07500 encoded an B-1,3 endoglucanase. Conclusion: Time course transcriptional profiling of P. chrysogenum revealed several differentially expressed genes during mixed culture, potentially reflecting interactions between the eukaryotic and the prokaryotic systems. M-oM-^AM-!-1,3 endoglucanase produced by P. chrysogenum against B. subtilis signals may have application in improving the efficacy of antibiotics by degrading exopolysacchride biofilms of pathogenic bacteria. The objective of the present study is to investigate the response of P. chrysogenum to co-cultivation with B. subtilis. To trigger an interaction specific behaviour, steady state chemostat of P. chrysogenum Wisconsin 54-1255 was pulsed with B. subtilis. The dynamic, transcriptional and physiological responses of P. chrysogenum in mixed culture were monitored and analyzed. Several differentially expressed genes potentially reflected interactions between the eukaryotic and the prokaryotic systems. To test whether any bacterial signaling molecules are responsible for differential expression of selected fungal genes, P. chrysogenum cultures were inoculated with supernatant of B. subtilis culture, supernatants from mixed culture and with heat-inactivated B. subtilis. The specific transcriptional responses identified using microarray was verified by analysis of fermentation broth and functional characterization by expression of selected genes in S. cerevisiae.
2014-05-12 | E-GEOD-53286 | biostudies-arrayexpress
Project description:In microbial production of non-catabolic products, a loss of production capacity upon long-term cultivation (for example, chemostat), a phenomenon called strain degeneration, is nearly always observed. In this study, a systems biology approach (monitoring changes from gene to produced flux) was used to study degeneration of penicillin production by Penicillium chrysogenum in ethanol-limited chemostat fermentations where the biomass specific penicillin production rate decreased 10-fold within 30 generations. Results showed that the copy number of penicillin gene clusters and expression levels of central metabolism showed little decrease. With respect to penicillin production, major changes were observed: a strong downregulation of the cysteine pathway in agreement with its nearly 10-fold flux reduction. Also, levels of ACVS and IPNS, two penicillin pathway enzymes, and the penicillin transport capacity decreased many fold. This indicates that degeneration is caused by changed regulation of post-translational modifications or an increased protein degradation rate of these proteins. Continued subcultivation of a degenerated culture resulted in partial recovery of the biomass specific penicillin production rate, however, it was still 5-fold lower than the peak biomass specific penicillin production rate. Prolonged chemostat cultivations up to 500 hours (30 generations) with ethanol as the sole limiting carbon source were performed because degeneration is found to be more pronounced with ethanol as a limiting sole carbon source compared to glucose. Measurements included genome level (number of penicillin gene clusters), genome-wide transcriptome, protein levels of penicillin pathway enzymes, number of peroxisomes (microbodies where part of the penicillin pathway occurs), metabolome (of central metabolism, nucleotides, penicillin pathway intermediates including intracellular PAA and PenG) and fluxome.
2011-06-10 | E-GEOD-24212 | biostudies-arrayexpress
Project description:Thyroid carcinoma (TC) is generally associated with good prognosis, nevertheless no effective treatments are available for aggressive forms not cured by current therapies. We previously identified the coatomer protein complex zeta 1 (COPZ1), as a new putative therapeutic target for TC, since its depletion impairs the viability of tumor cells, leads to abortive autophagy, ER stress, unfolded protein response and apoptosis, and reduces the tumor growth of TC xenograft models. In this study, by combining genomic, proteomic and functional approaches, we provided evidence that COPZ1 silencing stimulates a type I IFN-mediated viral mimicry response, boosts the production of several inflammatory molecules and finally induces immunogenic cell death, which, in turn, promotes dendritic cell maturation and subsequent activation of T cells. Collectively, our findings support the notion that COPZ1 targeting can be exploited as a new strategy to kill cancer cells with the subsequent involvement of an anti-tumor immune response.
2020-03-05 | PXD014423 | Pride
Project description:A group of gram positive bacteria that share the characteristic of fermenting hexose sugars to lactic acid are generally referred to as lactic acid bacteria (LAB). Enterococcus faecalis is one of the widely studied LABs due to a multiutude of reasons. On the one hand, it plays an important role in dairy industry, being for example a starter in cheese cultures. On the other hand, it accounts for a large part of the infections caused by the LABs in hospital environments. During the past few years, it developed resistance against most of the major antibiotics. Here, in an attempt to study its adaptive metabolism, a glutamine synthetase mutant (∆glnA) of E. faecalis was subjected to pH shift and the results from the integrative analysis of its metabolic network were compared to those of the wild type. The proteome data generated in this study were used to constrain the genome-scale metabolic network at two pH level, aiming to reduce the solution space and improve the accuracy of model simulation. This data particularly helped to come up with a new design for the amino acid transport system in the genome-scale model, resulting in an accurate reproduction of the metabolic behaviour of E. faecalis.
2022-01-14 | PXD030778 | Pride
Project description:The present study aims to explore chemostat-based transcriptome analysis of mixed cultures by investigating interactions between the yeast S. cerevisiae and the lactic acid bacterium Lb. bulgaricus . S. cerevisiae and Lb. bulgaricus are both frequently encountered in kefir, a fermented dairy product (25). In the context of this study, this binary culture serves as a model for the many traditional food and beverage fermentation processes in which yeasts and lactic acid bacteria occur together (19,26-30). The design of the cultivation conditions was based on the observation that Lb. bulgaricus, but not S. cerevisiae, can use lactose as a carbon source for growth and that S. cerevisiae, but not Lb. bulgaricus, can grow on galactose that is released upon hydrolysis of lactose by the bacterial M-NM-2-galactosidase. Mixed populations of yeasts and lactic acid bacteria occur in many dairy, food and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the co-cultures, five mechanisms of interaction were identified. 1. Lb. bulgaricus hydrolyses lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. bulgaricus, is excreted and provides a carbon source for yeast. 2. In pure cultures, Lb. bulgaricus only grows at increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. 3. Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacteria. 4. A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. bulgaricus. 5. Transcriptome analysis of Lb. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipids metabolism suggesting either a competition of the two microorganisms for fatty acids, or a response to the ethanol produced by S. cerevisiae. To our knowledge, this is the first transcriptome study of a cross-kingdom binary mixed culture that analyses responses of both microorganisms. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigated microbial interaction in mixed populations. To investigate the impact of of co-cultivation with Lb. bulgaricus on S. cerevisiae, a DNA microarray-based transcriptome analysis of S. cerevisiae's response was performed on anaerobic, lactose-limited chemostat cultures grown in the presence and absence of L. bulgaricus.
2013-08-22 | E-GEOD-45776 | biostudies-arrayexpress
Project description:The hop plant, Humulus lupulus L., contains an exceptionally high content of secondary metabolites, the hop iso-α-acids, which possess a range of beneficial properties including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigates molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acids detoxification and tolerance. Further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves two major processes: active export of iso-α-acids across the plasma membrane and active proton pumping into the vacuole by the V-ATPase to enable vacuolar sequestration of iso-α-acids. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelator. Experiment Overall Design: Two complementary genome-wide approaches were employed to investigate cellular responses of S. cerevisiae to hop extracts enriched in iso-α-acids. Microarray transcriptome analysis was performed on chemostat cultures of an S. cerevisiae reference strain grown in the presence and absence of iso-α-acids. In addition, screening of the nearly complete set of yeast open reading frame (ORF) haploid knock-outs generated by the Saccharomyces Genome Deletion Project (SGDP) (Open Biosystems) identified the mutants with increased hop sensitivity. Subsequently, involvement of selected genes and cellular processes in hop acid sensitivity and tolerance was analyzed by construction and detailed analysis of selected mutant strains.
2009-11-28 | E-GEOD-15094 | biostudies-arrayexpress
Project description:Raw expression values (CHP data) for transcriptional profiling of the response of Saccharomyces cerevisiae to challenges with lactic acid at pH 3 and pH 5. Experiment Overall Design: The laboratory reference strain CEN.PK 113-7D (MATa) was grown at 30 °C in 1.5-L chemostat fermentors (Applikon, Schiedam, The Netherlands) with a working volume of 1-L using an electronic level sensor to maintain a constant volume. All cultures, including the reference, were fed with minimal medium as described by Verduyn et al. (1992) with 25 g L-1 glucose as the limiting nutrient and 0.15 ml L-1 silicone antifoam (BDH, Poole, England) to prevent excessive foaming. The dilution rate was set to 0.10 h-1 and the pH was controlled at 5.0 with the automatic addition (ADI 1031 bio controller, Applikon) of 2 M KOH. The stirrer speed was set at 800 RPM and anaerobicity was maintained by sparging the fermentor with N2 gas at 500 ml min-1. To prevent diffusion of oxygen, the fermentor was equipped with Norprene tubing and Viton O-rings and the medium vessel was also flushed with N2 gas. A comparable degree of weak acid uncoupling was ensured by decreasing the biomass yield to approximately 50% of the reference condition (no organic acids added) with the addition of the appropriate concentration of lactic acid to the reservoir media. Experiment Overall Design: Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications. Double-stranded cDNA synthesis was carried out using 15 μg of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix). The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix). Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 μg of biotinylated cRNA. Experiment Overall Design: Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0 and Data Mining Tool v3.0.
2008-10-20 | E-GEOD-10066 | biostudies-arrayexpress
Project description:Although protein acetylation is widely observed, it has been associated with few specific regulatory functions making it poorly understood. To interrogate its functionality, we analyzed the acetylome in Escherichia coli knockout mutants of cobB, the only known sirtuin-like deacetylase, and patZ, the best-known protein acetyltransferase. For four growth conditions, more than 2,000 unique acetylated peptides, belonging to 809 proteins, were identified and differentially quantified. Nearly 65% of these proteins are related to metabolism. The global activity of CobB contributes to the deacetylation of a large number of substrates and has a major impact on physiology. Apart from the regulation of acetyl-CoA synthetase, we found that CobB-controlled acetylation of isocitrate lyase contributes to the fine-tuning of the glyoxylate shunt. Acetylation of the transcription factor RcsB prevents DNA binding, activating flagella biosynthesis and motility, and increases acid stress susceptibility. Surprisingly, deletion of patZ increased acetylation in acetate cultures, which suggests that it regulates the levels of acetylating agents. The results presented offer new insights into functional roles of protein acetylation in metabolic fitness and global cell regulation. In this study we aimed to discover how the deregulation of protein acetylation could alter physiology in E. coli. We observed that the deletion of both cobB or patZ genes in E. coli altered gene expression, specially those genes related with motility, chemotaxis and acid stress response. We used three biological replicates in this study and each condition. The control in these experiments was E. coli BW25113
2014-10-07 | E-GEOD-62094 | biostudies-arrayexpress