Comparative transcriptome analysis of yeast expressing the fungal desaturases
ABSTRACT: To study how the presence of PUFAs influences central cellular processes, and in order to perform lipidome, transcriptome and molecular studies we decided to use yeast as a model organism. We therefore co-expressed Δ12-desaturase and Δ6- desaturase genes from Mucor rouxii in S. cerevisiae with the objective to obtain a yeast strain that contains PUFAs, especially linoleic acid (LA, C18:2Δ9,12) and γ-linolenic acid (GLA, C18:3Δ6,9,12), in its membranes. The resulting yeast strain was analyzed in details in order to quantify the fluxes through the fatty acid biosynthetic pathways and the genome-wide transcriptional response to production of LA and GLA. Overall design: Three recombinant yeasts were constructed and grown in glucose-limited condition. Steady-state cultures were harvested for RNA extraction and hybridization on Affymetrix microarrays. Comparative transcriptome analysis between S. cerevisiae recombinants expressing M. rouxii D12-desaturase (D12) and co-expressing D12-desaturase and D6-desaturase genes (D126) and the reference strain (REF) was implemented.
Project description:To study how the presence of PUFAs influences central cellular processes, and in order to perform lipidome, transcriptome and molecular studies we decided to use yeast as a model organism. We therefore co-expressed Δ12-desaturase and Δ6- desaturase genes from Mucor rouxii in S. cerevisiae with the objective to obtain a yeast strain that contains PUFAs, especially linoleic acid (LA, C18:2Δ9,12) and γ-linolenic acid (GLA, C18:3Δ6,9,12), in its membranes. The resulting yeast strain was analyzed in details in order to quantify the fluxes through the fatty acid biosynthetic pathways and the genome-wide transcriptional response to production of LA and GLA. Three recombinant yeasts were constructed and grown in glucose-limited condition. Steady-state cultures were harvested for RNA extraction and hybridization on Affymetrix microarrays. Comparative transcriptome analysis between S. cerevisiae recombinants expressing M. rouxii D12-desaturase (D12) and co-expressing D12-desaturase and D6-desaturase genes (D126) and the reference strain (REF) was implemented.
Project description:Experimental studies confirmed n-6 type polyunsaturated fatty acid as pro-carcinogenic factor and n-3 fatty acid as cancer restraining agent; though their mode of action on tumor cells are still unclear. To review the contrasting effect of omega-3 and omega-6 fatty acids over carcinoma by studying genomic alteration in treated cancer cells, two members from each family of PUFAs, namely EPA and DHA from n-3 PUFA family and, AA and LA from n-6 PUFA family was selected to treat four breast cancer cell lines: MDA-MB231, MDA-MB435S, HCC2218 and MCF-7 for two time period- 6hr and 24hr.
Project description:The effect of a complex cellular matrix extracted from yeast (S. cerevisiae, strain YSBN6 (MATa; genotype: FY3 ho::HphMX4 derived from the S288C parental strain)) on the degradation profiles of nucleotide triphosphates extracted under typical boiling ethanol conditions was evaluated.
Project description:During fermentation Saccharomyces yeast produces various aroma-active metabolites determining the different characteristics of aroma and taste in fermented beverages. Amino acid utilization by yeast during brewer´s wort fermentation is seen as linked to flavour profile. To better understand the relationship between the biosynthesis of aroma relevant metabolites and the importance of amino acids, DNA microarrays were performed for Saccharomyces cerevisiae strain S81 and Saccharomyces pastorianus var. carlsbergensis strain S23, respectively. Thereby, changes in transcription of genes were measured, which are associated with amino acid assimilation and its derived aroma-active compounds during fermentation. 48 samples were used in this experiment
Project description:The optimal ratio of omega-6 to omega-3 polyunsaturated fatty acids (PUFAs) is important for keeping homeostasis of biological processes and metabolism, yet the underlying biological mechanism is poorly understood. The objective of this study was to identify changes in the pig liver transcriptome induced by a diet enriched with omega-6 and omega-3 fatty acids, and to characterize the biological mechanisms related to PUFA metabolism. Polish Landrace pigs (n =12) were fed diet enriched with linoleic acid (LA, omega-6) and alpha-linolenic acid (ALA, omega-3 family) or standard diet as a control. The fatty acids profiling was assayed in order to verify how feeding influenced the fatty acids content in liver, and subsequently next-generation sequencing (NGS) was used to identify differentially expressed genes (DEG) between transcriptomes between dietary groups. The biological mechanisms and pathway interaction networks were identified by analysis in DAVID and Cytoscape tools. Fatty acids profile analysis indicated a higher contribution of PUFAs in liver for LA and ALA-enriched diet group, particularly for the omega-3 fatty acids family, but not omega-6. Next-generation sequencing identified 3,565 DEG, 1,484 of which were induced and 2,081 were suppressed by PUFA supplemenation. Low ratio of omega-6/-3 fatty acids resulted in modulation of fatty acids metabolism pathways and over-representation of genes involved in membrane composition, signal transduction and immune response pathways. In conclusion, a diet enriched with omega-6 and omega-3 fatty acids altered the transcriptomic profile of the pig liver and affected a set of genes involved in metabolic pathways important to animal health status. Hepatic mRNA profiles of Polish Landrace pig breed fed two different diets, were generated by deep sequencing, using Illumina MiSeq. Experimental diet was enriched with polyunsaturated fatty acids (omega-6 and omega-3), while standard diet remain as a cotrol. 2 pooled samples each containing RNA extracts from 6 individuals livers were analyzed.
Project description:We designed and experimentally validated an in silico gene deletion strategy for engineering endogenous one-carbon (C1) metabolism in yeast. Specifically, a Flux Balance Analysis (FBA) model predicted that five genes ALT2, FDH1, FDH2, FUM1, and ZWF1, when deleted in combination, would cause Saccharomyces cerevisiae to overproduce and secrete formic acid under aerobic growth conditions. Once constructed, the quintuple mutant strain showed the predicted increase in formic acid secretion relative to a formate dehydrogenase mutant (fdh1 fdh2 ), while formic acid secretion in wild-type yeast was undetectable. Gene expression and physiological data generated post hoc identified a mitochondrial deficiency phenotype in the engineered strain and regulatory events that suggest a role for multisite modulation—the coordinated expression of multiple pathway enzymes—in controlling yeast C1 metabolism. Together, these results demonstrate that FBA-based modeling strategies can be useful tools for non-fermentative pathway design and discovery in eukaryotic microbes. In addition, the results indicate that seemingly unrelated mutations can be combined to modulate flux through biochemical reactions that interact at a systems level across subcellular compartments. Overall design: Samples were processed in biological triplicate with one sample processed with dye orientations reversed to minimize the effects of incorporation bias.
Project description:The sre1 pathway senses molecular oxygen in the fission yeast, Schizosaccharomyces pombe. When oxygen becomes limiting, Sre1 is trafficked from the ER to the Golgi where it is cleaved through the actions of the 5-member Dsc E3 ligase complex. The released N-terminus then upregulates genes required for adaptation to hypoxia. Although the sre1 pathway is responsible for regulating a majority of the genes required for hypoxic growth, it is evident that other regulatory genes are involved in this response. To identify novel regulators of adaptation to hypoxia, we screened the S. pombe nonessential haploid deletion collection. This genome wide screen identified nine genes required for production of the membrane-bound transcription factor Sre1N. The characterization of one of these, the S. pombe transcriptional activator mga2, is presented. mga2 is the homolog of SPT23 and MGA2 in S. cerevisiae. In S. cerevisiae SPT23 and MGA2 regulate the transcription of genes involved in lipid metabolism, including the delta-9 fatty acid desaturase, OLE1. We show S. pombe mga2 also regulates lipid metabolism, and that in the absence of mga2, the lipidome is disrupted. Supplementation with unsaturated fatty acids rescued Sre1 cleavage in mga2Δ cells. However, mga2 is not simply required for activation of Sre1 as mga2Δ cells have a normoxia growth defect that is unrelated to Srel activity. Loss of mga2 results in aberrant Dsc1 glycosylation, suggesting that the Dsc complex is not properly localized to the Golgi. These results establish unsaturated fatty acids as a regulator of the SREBP pathway in fission yeast. Overall design: 4 samples analyzed in 2 comparisons: 2 biological replicates of each WT (sre1D) and mga2D KO (sre1D/mga2D) in a dye-swap configuration.
Project description:Aim: Analyse inhibitory effects of galacturonic acid, an important constituent of plant biomass hydrolysates, on growing and starving cultures of Saccharomyces cerevisiae CEN.PK113-7D. Method & Results: Biomass yields in aerobic and anaerobic glucose-limited chemostat cultures (pH 3.5) were reduced by 25 and 10%, respectively, upon addition of 10 g∙l-1 galacturonic acid. Genes previously reported to show a transcriptional response to other organic acids were overrepresented in a set of galacturonic-acid responsive genes identified by microarray analysis. These results suggested that galacturonic acid causes weak-acid uncoupling of the yeast plasma membrane pH gradient. Consistent with this hypothesis, galacturonate-accelerated loss of viability in starving cell suspensions was strongly pH dependent. Loss of viability was much slower in a strain in which all HXT (hexose transporter) genes were deleted. Moreover, deletion of HXT genes alleviated growth inhibition on ethanol observed at galacturonic acid concentrations of 10 g∙l-1 and above. Conclusions: At low pH, galacturonic acid negatively affects the physiology of S. cerevisiae. Reduced sensitivity of hexose-transporter mutants indicated that one or more HXT transporters are involved in transport of galacturonic acid. Significance and Impact: This study shows that galacturonic acid toxicity should be taken into account in process development for yeast-based fermentative conversion of pectin-rich feedstocks such as sugar beet pulp and citrus peel. Involvement of hexose transporters in galacturonic acid toxicity provides leads for improving tolerance. To investigate the impact of galacturonic acid on S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic, glucose-limited chemostat cultures grown in the presence and absence of 10 g∙l-1 galacturonic acid at pH3.5.
Project description:The yeast Saccharomyces cerevisiae is an important component of the wine fermentation process and determines various attributes of the final product. However, lactic acid bacteria (LAB) are also an integral part of the microflora of any fermenting must. Various wine microorganism engineering projects have been endeavoured in the past in order to change certain wine characteristics, namely aroma compound composition, ethanol concentration, levels of toxic/ allergenic compounds etc. Most of these projects focus on a specific gene or pathway, whereas our approach aims to understand the genetically complex traits responsible for these phenotypes in a systematic manner by implementing a transcriptomic analysis of yeast in mixed fermentations with the LAB O. oeni. Our aim is to investigate interactions between yeast and LAB on a gene expression level to identify targets for modification of yeast and O. oeni in a directed manner. Our goal was to identify the impact that the common wine microorganism O. oeni (malolactic bacteria) has on fermenting yeast cells on a gene expression level. To this end we co-inoculated the yeast and bacteria at the start of fermentation in a synthetic wine must, using yeast-only fermentations witout O. oeni as a control. Overall design: Fermentations were carried out in synthetic wine must in triplicate for both the control S. cerevisiae VIN13 strain and the mixed fermentation of VIN13 and O. oeni (strain S5). Sampling of yeast for RNA extractions were performed at day 3 of fermentation, during the exponential growth phase of the yeast cells, and again at day 7 of fermentation, during the early stationary growth phase.
Project description:Production of D-xylonate in the yeast S. cerevisiae represents an example of bioprocess development for more sustainable production of value-added chemicals from cheap raw material or waste. Previously it was shown that the production of D-xylonate led to its significant intracellular accumulation and to dramatic loss of viability during the production process. In order to identify the physiological or pathological responses associated with D-xylonate production, we performed a time-course transcriptome analysis of D-xylonate production in yeast cultivated in a bioreactor. Comparison of the transcriptomes of D-xylonate producing strain with control strain showed considerably higher expression in the xylonate producing strain of the genes controlled by the cell wall integrity pathway (CWI) and of some genes previously identified as upregulated in response to the organic acid stress. Surprisingly, also genes encoding proteins involved in translation, ribosome structure and RNA metabolism – the processes commonly found to be down-regulated under virtually every condition causing cellular stress – were upregulated during the D-xylonate production. The overall transcriptional responses were, therefore, very dissimilar to those previously reported as being associated with diverse stresses including the organic acid treatment and production. In addition, it was observed that the consumption of ethanol was slower and the level of trehalose was lower in the D-xylonate producing strain. Validation experiments including Slt2 kinase phosphorylation profiles and the quantitative PCR analyses of selected gene showed remarkably good match with our findings and confirmed the observations made in the transcriptome analysis. The production of organic acids has a major impact on the physiology of yeast cells. There is, however, very limited overlap at the transcriptional level in responses to treatment or production of different acids. The loss of viability, observed during production and accumulation of D-xylonate, seems to be caused by erroneous interpretation of environmental signals causing a failure in entering the stationary phase and eventually leading to depletion of scarce resources by the affected cells. This, together with intracellular acidification, inevitably results in cell death. Overall design: The study compares two yeast strains, a D-xylonate producing strain, expressing XylB gene from C. crescentus, and the isogenic parental strain, an industrial hydrolysate tolerant S. cerevisiae strain B67002. The strains were grown in bioreactors and samples for microarrays were taken from 6 time points during the cultivation (0h, 7h, 23h, 47h, 49h and 71h). Three replicate cultures were performed for each strain. The total number of microarrays is 36. Each arrays measures 5,777 genes from Saccharomyces cerevisiae with eight 60-mer probes per gene, with three-fold technical redundancy.