| 1. |
- Nookaew, Intawat, 1977, et al.
(författare)
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A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays : a case study in Saccharomyces cerevisiae
- 2012
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:20, s. 10084-10097
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Tidskriftsartikel (refereegranskat)abstract
- RNA-seq, has recently become an attractive method of choice in the studies of transcriptomes, promising several advantages compared with microarrays. In this study, we sought to assess the contribution of the different analytical steps involved in the analysis of RNA-seq data generated with the Illumina platform, and to perform a cross-platform comparison based on the results obtained through Affymetrix microarray. As a case study for our work we, used the Saccharomyces cerevisiae strain CEN.PK 113-7D, grown under two different conditions (batch and chemostat). Here, we asses the influence of genetic variation on the estimation of gene expression level using three different aligners for read-mapping (Gsnap, Stampy and TopHat) on S288c genome, the capabilities of five different statistical methods to detect differential gene expression (baySeq, Cuffdiff, DESeq, edgeR and NOISeq) and we explored the consistency between RNA-seq analysis using reference genome and de novo assembly approach. High reproducibility among biological replicates (correlation >= 0.99) and high consistency between the two platforms for analysis of gene expression levels (correlation >= 0.91) are reported. The results from differential gene expression identification derived from the different statistical methods, as well as their integrated analysis results based on gene ontology annotation are in good agreement. Overall, our study provides a useful and comprehensive comparison between the two platforms (RNA-seq and microrrays) for gene expression analysis and addresses the contribution of the different steps involved in the analysis of RNA-seq data.
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| 2. |
- Papini, Marta, 1981, et al.
(författare)
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Phosphoglycerate mutase knock-out mutant Saccharomyces cerevisiae: Physiological investigation and transcriptome analysis
- 2010
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 5:10, s. 1016-1027
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is able to adapt its metabolism to grow on different carbon sources and to shift to non-fermentative growth on C-2 or C-3 carbon sources (ethanol, acetate, or glycerol) through the activation of gluconeogenesis. Here, we studied the response to tine deletion of the glycolytic and gluconeogenic gene GPM1, encoding for phosphoglycerate mutase. It was previously shown that a S. cerevisiae strain with non-functional copies of GPM1 can only grow when glycerol and ethanol are both present as carbon sources, whilst addition of glucose was shown to strongly inhibit growth. It was suggested that glycerol is needed to feed gluconeogenesis whilst ethanol is required for respiration. Here, we studied the physiological response of the GPM1 knock-out mutant through fermentation and transcriptome analysis. Furthermore, we compared the physiological results with those obtained through simulations using a genome-scale metabolic model, showing that glycerol is only needed in small amounts for growth. Our findings strongly suggest a severely impaired growth ability of the knock-out mutant, which presents increased transcript levels of genes involved in the pentose phosphate pathway and in the glyoxylate shunt. These results indicate an attempt to compensate for the energy imbalance caused by the deletion of the glycolytic/gluconeogenic gene within the mutant.
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| 3. |
- Chen, Yun, 1978, et al.
(författare)
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Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production
- 2012
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 12:5, s. 598-607
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Tidskriftsartikel (refereegranskat)abstract
- 2 mu m-based episomal expression vectors are widely used in Saccharomyces cerevisiae for recombinant protein production and synthetic pathway optimization. In this study, we report a new approach to increase the plasmid copy number (PCN) and thus improve the expression of plasmid-encoded proteins. This was achieved by combining destabilization of the marker protein with decreasing the marker gene transcription level. Destabilization of the marker protein alone by fusing a ubiquitin/N-degron tag (ubi-tag) to the N-terminus of the Ura3 marker protein could increase the PCN and activity of LacZ expressed from the same vector. When arginine was exposed at the N-terminus of the marker protein after cleavage of ubiquitin, the PCN and LacZ activity were increased by 7080%. Replacement of the native URA3 promoter with the HXT1, KEX2 or URA3-d promoter resulted in an increase in the PCN and LacZ activity by about 30100%. Combining the ubi-tag and promoter modification of the marker gene, increased the PCN and LacZ activity by threefold. We also demonstrated that this new expression vectors can be used to increase enzyme activity by improving patchoulol production by threefold.
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| 4. |
- Hou, J., et al.
(författare)
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Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae
- 2010
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 76:3, s. 851-859
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Tidskriftsartikel (refereegranskat)abstract
- Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD(+)-dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.
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| 5. |
- Scalcinati, Gionata, 1981, et al.
(författare)
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Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae
- 2012
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 11, s. Article Number: 117-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundSesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of sesquiterpene production via extraction from plants or chemical synthesis there is interest in developing alternative and cost efficient bioprocesses. The hydrocarbon α-santalene is a precursor of sesquiterpenes with relevant commercial applications. Here, we construct an efficient Saccharomyces cerevisiae cell factory for α-santalene production.ResultsA multistep metabolic engineering strategy targeted to increase precursor and cofactor supply was employed to manipulate the yeast metabolic network in order to redirect carbon toward the desired product. To do so, genetic modifications were introduced acting to optimize the farnesyl diphosphate branch point, modulate the mevalonate pathway, modify the ammonium assimilation pathway and enhance the activity of a transcriptional activator. The approach employed resulted in an overall α-santalene yield of a 0.0052 Cmmol (Cmmol glucose)-1 corresponding to a 4-fold improvement over the reference strain. This strategy, combined with a specifically developed continuous fermentation process, led to a final α-santalene productivity of 0.036 Cmmol (g biomass)-1 h-1.ConclusionsThe results reported in this work illustrate how the combination of a metabolic engineering strategy with fermentation technology optimization can be used to obtain significant amounts of the high-value sesquiterpene α-santalene. This represents a starting point toward the construction of a yeast “sesquiterpene factory” and for the development of an economically viable bio-based process that has the potential to replace the current production methods.
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| 6. |
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| 7. |
- Scalcinati, Gionata, 1981, et al.
(författare)
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Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption
- 2012
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 12:5, s. 582-597
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Tidskriftsartikel (refereegranskat)abstract
- Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S similar to cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S similar to cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S similar to cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source.
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| 8. |
- Scalcinati, Gionata, 1981
(författare)
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Metabolic Engineering of Saccharomyces cerevisiae for Sesquiterpene Production
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Industrial biotechnology aims to develop robust “microbial cell factories”, to produce an array of added value chemicals presently dominated by petrochemical processes. The exploitation of an efficient microbial production as sustainable technology has an important impact for our society. Sesquiterpenes are a class of natural products with a diverse range of attractive industrial proprieties. Due to economic difficulties of their production via traditional extraction processes or chemical synthesis there is interest in developing alternative and cost efficient bio-processes. Microbial cells engineered for efficient production of plant sesquiterpenes may allow for a sustainable and scalable production of these compounds. Saccharomyces cerevisiae is one of the most robust and characterized microbial platforms suitable to be exploited for bio-production. The hydrocarbon α-santalene is a precursor of sesquiterpenes with relevant commercial application and was selected as case study. Here, for the first time a S. cerevisiae strain capable of producing high levels of α-santalene was constructed through a multidisciplinary system level metabolic engineering approach. First, a minimal engineering approach was applied to address the feasibility of α-santalene production in S. cerevisiae. Successively, a rationally designed metabolic control strategy with the aim to dynamically modulate a key metabolic step to achieve optimal sesquiterpene production was applied, combined with the engineering of the main regulatory checkpoint of targeted pathway. It was possible to divert the carbon flux toward the sesquiterpene compound, and the resulting strain shows a 88-fold improvement in α-santalene productivity. A second round of strain optimization was performed using a multistep strategy focused to increase precursors and co-factor supply to manipulate the yeast metabolic network in order to further redirect the carbon toward the desired product. This approach results in an overall increase of 1.9-fold in α-santalene productivity. Furthermore, strain improvement was integrated with the development of an efficient fermentation/ downstream recovery process, resulting in a 1.4-fold improvement in productivity and a final α-santalene titer of 193 mg l-1. Finally, the substrate utilization range of the selected platform was expanded to use xylose as alternative carbon source for biorefinery compatibility, via pathway reconstruction and an evolutionary strategy approach, resulting in a strain capable of rapid growth and fast xylose consumption. The results obtained illustrate how the synergistic application of multilevel metabolic engineering and bioprocess engineering can be used to obtain a significant amount of high value sesquiterpene in yeast. This represents a starting point toward the construction of a yeast “sesquiterpene production factory” and for the development of an economically viable bio-based process that has the potential to replace the current production methods.
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| 9. |
- Tippmann, Stefan, 1986, et al.
(författare)
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Production of farnesene and santalene by Saccharomyces cerevisiae using fed-batch cultivations with RQ-controlled feed
- 2016
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 113:1, s. 72-81
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Tidskriftsartikel (refereegranskat)abstract
- Terpenes have various applications as fragrances, cosmetics and fuels. One of the most prominent examples is the sesquiterpene farnesene, which can be used as diesel substitute in its hydrogenated form farnesane. Recent metabolic engineering efforts have enabled efficient production of several terpenes in Saccharomyces cerevisiae and Escherichia coli. Plant terpene synthases take on an essential function for sesquiterpene production as they catalyze the specific conversion of the universal precursor farnesyl diphosphate (FPP) to the sesquiterpene of interest and thereby impose limitations on the overall productivity. Using farnesene as a case study, we chose three terpene synthases with distinct plant origins and compared their applicability for farnesene production in the yeast S. cerevisiae. Differences regarding the efficiency of these enzymes were observed in shake flask cultivation with maximal final titers of 4mg/L using -farnesene synthase from Malus domestica. By employing two existing platform strains optimized for sesquiterpene production, final titers could be raised up 170mg/L in fed-batch fermentations with RQ-controlled exponential feeding. Based on these experiments, the difference between the selected synthases was not significant. Lastly, the same fermentation setup was used to compare these results to production of the fragrance sesquiterpene santalene, and almost equivalent titers were obtained with 163mg/L, using the highest producing strain expressing a santalene synthase from Clausena lansium. However, a reduction of the product yield on biomass by 50% could indicate a higher catalytic efficiency of the farnesene synthase. Biotechnol. Bioeng. 2016;113: 72-81. (c) 2015 Wiley Periodicals, Inc.
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