Transcriptome-wide expression profile of yeast under different carbon sources
ABSTRACT: Carbon source is the basic nutrition and is essential for yeast growth. We grew the yeast cells (BY4741 strain) under different carbon sources including glucose with different concentration, galactose and raffinose. We generated bulk-cell RNA-seq data and investigated the dynamics of gene expression profiles under different growth conditions. We also generated single-cell RNA-seq data for yeast cells under 2% glucose, and explored the heterogeneity of gene expression within a cell population. Overall design: Saccharomyces cerevisiae BY4741 strain was used. The bulk-cell RNA-seq and single-cell RNA-seq data were generated for yeast cells under different growth media.
Project description:<h4>Background</h4>The model yeast<i>, Saccharomyces cerevisiae</i>, is not known to be oleaginous. However, an industrial wild-type strain, D5A, was shown to accumulate over 20% storage lipids from glucose when growth is nitrogen-limited compared to no more than 7% lipid accumulation without nitrogen stress.<h4>Methods and results</h4>To elucidate the mechanisms of <i>S. cerevisiae</i> D5A oleaginicity, we compared physiological and metabolic changes; as well as the transcriptional profiles of the oleaginous industrial strain, D5A, and a non-oleaginous laboratory strain, BY4741, under normal and nitrogen-limited conditions using analytic techniques and next-generation sequencing-based RNA-Seq transcriptomics. Transcriptional levels for genes associated with fatty acid biosynthesis, nitrogen metabolism, amino acid catabolism, as well as the pentose phosphate pathway and ethanol oxidation in central carbon (C) metabolism, were up-regulated in D5A during nitrogen deprivation. Despite increased carbon flux to lipids, most gene-encoding enzymes involved in triacylglycerol (TAG) assembly were expressed at similar levels regardless of the varying nitrogen concentrations in the growth media and strain backgrounds. Phospholipid turnover also contributed to TAG accumulation through increased precursor production with the down-regulation of subsequent phospholipid synthesis steps. Our results also demonstrated that nitrogen assimilation via the glutamate-glutamine pathway and amino acid metabolism, as well as the fluxes of carbon and reductants from central C metabolism, are integral to the general oleaginicity of D5A, which resulted in the enhanced lipid storage during nitrogen deprivation.<h4>Conclusion</h4>This work demonstrated the disequilibrium and rebalance of carbon and nitrogen contribution to the accumulation of lipids in the oleaginous yeast <i>S. cerevisiae</i> D5A. Rather than TAG assembly from acyl groups, the major switches for the enhanced lipid accumulation of D5A (i.e., fatty acid biosynthesis) are the increases of cytosolic pools of acetyl-CoA and NADPH, as well as alternative nitrogen assimilation.
Project description:This experiment describes the transcriptional response to oleic acid as a sole carbon source of yeast strains differing in their respiratory competence: the BY4741 wild type strain is considered as normal, sco1 knock out mutant is respiratory deficient and sco2 knock out mutant has a better respiratory competence then the wild type strain. The three strains underwent metabolic shift from 2% glucose as a carbon source to oleic acid at both standard (0.1%) and high (5%) concentration. The transcriptional profiling comprises two time points after metabolic shift: 2 hours and 5 hours. mRNA levels were compared with reference growth condition: exponential phase in 2% glucose. The purpose of this experiment is to gain deeper insight in the relationship between fatty acid metabolism and respiratory metabolism.
Project description:Successful fermentations to produce ethanol require microbial strains that have a high tolerance to glucose and ethanol. Enhanced glucose/ethanol tolerance of the laboratory yeast Saccharomyces cerevisiae strain BY4741 under certain growth conditions as a consequence of the expression of a dominant mutant allele of the SPT15 gene (SPT15-300) corresponding to the three amino acid changes F177S, Y195H, and K218R has been reported (H. Alper, J. Moxley, E. Nevoigt, G. R. Fink, and G. Stephanopoulos, Science 314:1565-1568, 2006). The SPT15 gene codes for the TATA-binding protein. This finding prompted us to examine the effect of expression of the SPT15-300 allele in various yeast species of industrial importance. Expression of SPT15-300 in leucine-prototrophic strains of S. cerevisiae, Saccharomyces bayanus, or Saccharomyces pastorianus (lager brewing yeast), however, did not improve tolerance to ethanol on complex rich medium (yeast extract-peptone-dextrose). The enhanced growth of the laboratory yeast strain BY4741 expressing the SPT15-300 mutant allele was seen only on defined media with low concentrations of leucine, indicating that the apparent improved growth in the presence of ethanol was indeed associated with enhanced uptake and/or utilization of leucine. Reexamination of the microarray data published by Alper and coworkers likewise suggested that expression of genes coding for the leucine permeases, Tat1p and Bap3p, were upregulated in the SPT15-300 mutant, as was expression of the genes ARO10, ADH3, ADH5, and SFA1, involved in leucine degradation.
Project description:Xanthophyllomyces dendrorhous is a carotenogenic yeast with a singular metabolic capacity to produce astaxanthin, a valuable antioxidant pigment. This yeast can assimilate several carbon sources and sustain fermentation even under aerobic conditions. Since astaxanthin biosynthesis is affected by the carbon source, the study of carotenogenesis regulatory mechanisms is key for improving astaxanthin yield in X. dendrorhous This study aimed to elucidate the regulation of the metabolism of different carbon sources and the phenomenon of catabolic repression in this yeast. To this end, protein and transcript levels were quantified by iTRAQ (isobaric tags for relative and absolute quantification) and transcriptomic sequencing (RNA-seq) in the wild-type strain under conditions of glucose, maltose, or succinate treatment and in the mutant strains for genes MIG1, CYC8, and TUP1 under conditions of glucose treatment. Alternative carbon sources such as maltose and succinate affected the relative abundances of 14% of the wild-type proteins, which were mainly grouped into the carbohydrate metabolism category, with the glycolysis/gluconeogenesis and citrate cycle pathways being the most highly represented pathways. Each mutant strain showed significant proteomic profile changes, affecting approximately 2% of the total proteins identified, compared to the wild-type strain under glucose treatment conditions. Similarly to the results seen with the alternative carbon sources, the changes in the mutant strains mainly affected carbohydrate metabolism, with glycolysis/gluconeogenesis and the pentose phosphate and citrate cycle pathways being the most highly represented pathways. Our results showed convergence between carbon assimilation and catabolic repression in the strains studied. Interestingly, indications of cooperative, opposing, and overlapping processes during catabolic regulation were found. We also identified target proteins of the regulatory processes, reinforcing the likelihood of catabolic repression at the posttranscriptional level.IMPORTANCE The conditions affecting catabolic regulation in X. dendrorhous are complex and suggest the presence of an alternative mechanism of regulation. The repressors Mig1, Cyc8, and Tup1 are essential elements for the regulation of the use of glucose and other carbon sources. All play different roles but, depending on the growth conditions, can work in convergent, synergistic, and complementary ways to use carbon sources and to regulate other targets for yeast metabolism. Our results reinforced the belief that further studies in X. dendrorhous are needed to clarify a specific regulatory mechanism at the domain level of the repressors as well as its relationship with those of other metabolic repressors, i.e., the stress response, to elucidate carotenogenic regulation at the transcriptomic and proteomic levels in this yeast.
Project description:A880 has pop2 deletion and a very slow-growth phenotype with glycerol as carbon source. A880 transformed with 2-micron plasmids encoding STM1 can grow robustly on glycerol plates. The Arg237 on Stm1p can be methylated by Hmt1p. A880 transformed with 2-micron plasmids encoding the Stm1p R237K mutant retain the slow-growth phenotype on glycerol plates. We used microarrays to assess the transcription profiles of A880, and A880 transformed with STM1 or STM1 R237K mutant. The goal is to identify genes that are up- or down-regulated in the presence of STM1 R237K mutant but not in the presence of the wild type STM1. BY4741 (wild type control), A880 (BY4741 with pop2 deletion), A880STM1 (A880 transformed with STM1) and A880STM1K (A880 transformed with STM1 carrying an Arg237 to lysine substitution) cells were grown in SC medium with glucose as carbon source to early log phase. The cells were shifted to SC medium containing 2% glycerol and incubated for 12 hours before harvesting. Total RNAs were extracted for microarray analysis. Two data sets were generated from separate experiments and cell cultures.
Project description:Autophagy is a conserved intracellular degradation system in eukaryotes. Recent studies have revealed that autophagy can be induced not only by nitrogen starvation but also by many other stimuli. However, questions persist regarding the types of conditions that induce autophagy, as well as the particular kinds of autophagy that are induced under these specific conditions. In experimental studies, abrupt nutrient changes are often used to induce autophagy. In this study, we investigated autophagy induction in batch culture on low-glucose medium, in which growth of yeast (Saccharomyces cerevisiae) cells is clearly reflected exclusively by carbon source state. In this medium, cells pass sequentially through three stages: glucose-utilizing, ethanol-utilizing, and ethanol-depleted phases. Using GFP cleavage assay by immunoblotting methods, fluorescence microscopy, and transmission electron microscopy ultrastructural analysis, we found that bulk autophagy and endoplasmic reticulum-phagy are induced starting at the ethanol-utilizing phase, and bulk autophagy is activated to a greater extent in the ethanol-depleted phase. Furthermore, we found that mitophagy is induced by ethanol depletion. Microautophagy occurred after glucose depletion and involved incorporation of cytosolic components and lipid droplets into the vacuolar lumen. Moreover, we observed that autophagy-deficient cells grow more slowly in the ethanol-utilizing phase and exhibit a delay in growth resumption when they are shifted to fresh medium from the ethanol-depleted phase. Our findings suggest that distinct types of autophagy are induced in yeast cells undergoing gradual changes in carbon source availability.
Project description:Glycerol is considered as a promising substrate for biotechnological applications and the non-conventional yeast Yarrowia lipolytica has been used extensively for the valorization of this compound. Contrary to S. cerevisiae, Y. lipolytica seems to prefer glycerol over glucose and it has been reported previously that the presence of glycerol can suppress the consumption of glucose in co-substrate fermentations. Based on these observations, we hypothesized glycerol repression-like effects in Y. lipolytica, which are converse to well described carbon repression mechanisms ensuring the prioritized use of glucose (e.g., in S. cerevisiae). We therefore aimed to investigate this effect on the level of transcription. Strains varying in the degree of glucose suppression were chosen and characterized in high-resolution growth screenings, resulting in the detection of different growth phenotypes under glycerol-glucose mixed conditions. Two strains, IBT and W29, were selected and cultivated in chemostats using glucose, glycerol and glucose/glycerol as carbon sources, followed by an RNA-Seq-based transcriptome analysis. We could show that several transporters were significantly higher expressed in W29, which is potentially related to the observed physiological differences. However, most of the expression variation between the strains were regardless of the carbon source applied, and cross-comparisons revealed that the strain-specific carbon source responses underwent in the opposite direction. A deeper analysis of the substrate specific carbon source response led to the identification of several differentially expressed genes with orthologous functions related to signal transduction and transcriptional regulation. This study provides an initial investigation on potentially novel carbon source regulation mechanisms in yeasts.
Project description:Eat1 is a recently discovered alcohol acetyltransferase responsible for bulk ethyl acetate production in yeasts such as Wickerhamomyces anomalus and Kluyveromyces lactis These yeasts have the potential to become efficient bio-based ethyl acetate producers. However, some fundamental features of Eat1 are still not understood, which hampers the rational engineering of efficient production strains. The cellular location of Eat1 in yeast is one of these features. To reveal its location, Eat1 was fused with yeast-enhanced green fluorescent protein (yEGFP) to allow intracellular tracking. Despite the current assumption that bulk ethyl acetate production occurs in the yeast cytosol, most of Eat1 localized to the mitochondria of Kluyveromyces lactis CBS 2359 ?ku80 We then compared five bulk ethyl acetate-producing yeasts in iron-limited chemostats with glucose as the carbon source. All yeasts produced ethyl acetate under these conditions. This strongly suggests that the mechanism and location of bulk ethyl acetate synthesis are similar in these yeast strains. Furthermore, an in silico analysis showed that Eat1 proteins from various yeasts were mostly predicted as mitochondrial. Altogether, it is concluded that Eat1-catalyzed ethyl acetate production occurs in yeast mitochondria. This study has added new insights into bulk ethyl acetate synthesis in yeast, which is relevant for developing efficient production strains.IMPORTANCE Ethyl acetate is a common bulk chemical that is currently produced from petrochemical sources. Several Eat1-containing yeast strains naturally produce large amounts of ethyl acetate and are potential cell factories for the production of bio-based ethyl acetate. Rational design of the underlying metabolic pathways may result in improved production strains, but it requires fundamental knowledge on the function of Eat1. A key feature is the location of Eat1 in the yeast cell. The precursors for ethyl acetate synthesis can be produced in multiple cellular compartments through different metabolic pathways. The location of Eat1 determines the relevance of each pathway, which will provide future targets for the metabolic engineering of bulk ethyl acetate production in yeast.
Project description:Our previous results (Calafi et al 2020; PMID: 32339526) demonstrated a defect in cell proliferation when PPZ1 was overexpressed under the control of a tetO-based promoter that was, up to some extent, dependent of the availability of glucose. To further investigate this phenomenon, we have determined the transcriptional response triggered by the shift form high to low glucose, or to galactose, in normal cells and in cells overexpressing PPZ1 from the tetO7–driven multicopy pCM190 plasmid. Our data suggest that, under these circumstances, overexpression of Ppz1 interferes with the proper activation of a set of genes mainly involved in carbon metabolism, thus impairing the normal transcriptional adaptation to conditions in which glucose becomes limiting. Overall design: Two different types of BY4741-based yeast cells were used: 1) cells containing the empty pCM190 plasmid and 2) cells carrying the same pCM190 plasmid with the PPZ1 coding region under control of the doxycycline-repressed tetO7 promoter. Both types of cells were shifted to medium containing 2% glucose, 0.25% glucose or 2% galactose, and grown for 1h before sample collection.
Project description:BACKGROUND:Optimal glucose metabolism is central to the growth and development of cells. In microbial eukaryotes, carbon catabolite repression (CCR) mediates the preferential utilization of glucose, primarily by repressing alternate carbon source utilization. In fission yeast, CCR is mediated by transcriptional repressors Scr1 and the Tup/Ssn6 complex, with the Rst2 transcription factor important for activation of gluconeogenesis and sexual differentiation genes upon derepression. Through genetic and genome-wide methods, this study aimed to comprehensively characterize CCR in fission yeast by identifying the genes and biological processes that are regulated by Scr1, Tup/Ssn6 and Rst2, the core CCR machinery. RESULTS:The transcriptional response of fission yeast to glucose-sufficient or glucose-deficient growth conditions in wild type and CCR mutant cells was determined by RNA-seq and ChIP-seq. Scr1 was found to regulate genes involved in carbon metabolism, hexose uptake, gluconeogenesis and the TCA cycle. Surprisingly, a role for Scr1 in the suppression of sexual differentiation was also identified, as homothallic scr1 deletion mutants showed ectopic meiosis in carbon and nitrogen rich conditions. ChIP-seq characterised the targets of Tup/Ssn6 and Rst2 identifying regulatory roles within and independent of CCR. Finally, a subset of genes bound by all three factors was identified, implying that regulation of certain loci may be modulated in a competitive fashion between the Scr1, Tup/Ssn6 repressors and the Rst2 activator. CONCLUSIONS:By identifying the genes directly and indirectly regulated by Scr1, Tup/Ssn6 and Rst2, this study comprehensively defined the gene regulatory networks of CCR in fission yeast and revealed the transcriptional complexities governing this system.