| 1. |
- Hong, Kuk-ki, 1976, et al.
(författare)
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Unravelling evolutionary strategies of yeast for improving galactose utilization through integrated systems level analysis
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:29, s. 12179-12184
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Tidskriftsartikel (refereegranskat)abstract
- Identification of the underlying molecular mechanisms for a derived phenotype by adaptive evolution is difficult. Here, we performed a systems-level inquiry into the metabolic changes occurring in the yeast Saccharomyces cerevisiae as a result of its adaptive evolution to increase its specific growth rate on galactose and related these changes to the acquired phenotypic properties. Three evolved mutants (62A, 62B, and 62C) with higher specific growth rates and faster specific galactose uptake were isolated. The evolved mutants were compared with a reference strain and two engineered strains, SO16 and PGM2, which also showed higher galactose uptake rate in previous studies. The profile of intermediates in galactose metabolism was similar in evolved and engineered mutants, whereas reserve carbohydrates metabolism was specifically elevated in the evolved mutants and one evolved strain showed changes in ergosterol biosynthesis. Mutations were identified in proteins involved in the global carbon sensing Ras/PKA pathway, which is known to regulate the reserve carbohydrates metabolism. We evaluated one of the identified mutations, RAS2(Tyr112), and this mutation resulted in an increased specific growth rate on galactose. These results show that adaptive evolution results in the utilization of unpredicted routes to accommodate increased galactose flux in contrast to rationally engineered strains. Our study demonstrates that adaptive evolution represents a valuable alternative to rational design in bioengineering of improved strains and, that through systems biology, it is possible to identify mutations in evolved strain that can serve as unforeseen metabolic engineering targets for improving microbial strains for production of biofuels and chemicals.
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| 2. |
- Holm, A. K., et al.
(författare)
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Metabolic and Transcriptional Response to Cofactor Perturbations in Escherichia coli
- 2010
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:23, s. 17498-17506
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Tidskriftsartikel (refereegranskat)abstract
- Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli to lower the level of NADH and ATP, respectively. We used a global interaction network, comprising of protein interactions, transcriptional regulation, and metabolic networks, to integrate data from transcription profiles, metabolic fluxes, and the metabolite levels. We identified high-scoring networks for the two strains. The results revealed a smaller, but denser network for perturbations of ATP level, compared with that of NADH level. The action of many global transcription factors such as ArcA, Fnr, CRP, and IHF commonly involved both NADH and ATP, whereas others responded to either ATP or NADH. Overexpressing NADH oxidase invokes response in widespread aspects of metabolism involving the redox cofactors (NADH and NADPH), whereas ATPase has a more focused response to restore ATP level by enhancing proton translocation mechanisms and repressing biosynthesis. Interestingly, NADPH played a key role in restoring redox homeostasis through the concerted activity of isocitrate dehydrogenase and UdhA transhydrogenase. We present a reconciled network of regulation that illustrates the overlapping and distinct aspects of metabolism controlled by NADH and ATP. Our study contributes to the general understanding of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli.
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| 3. |
- Hou, Jin, 1982, et al.
(författare)
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Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae
- 2009
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 82:82, s. 909-919
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Tidskriftsartikel (refereegranskat)abstract
- During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.
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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. |
- Hou, J., et al.
(författare)
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Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae
- 2009
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:4-5, s. 253-261
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Tidskriftsartikel (refereegranskat)abstract
- Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation during metabolism. There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output. Therefore, cofactors are emerging to be attractive targets to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol production, while decreasing mitochondrial NADH lowered ethanol production. However, when these reactions were coupled with NADPH production, the metabolic changes were more moderated. The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations of the cofactors. The changes in product profile and intracellular metabolite levels were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation, as estimated from a simple stoichiometric model. The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications. (C) 2009 Elsevier Inc. All rights reserved.
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| 6. |
- Hou, J., et al.
(författare)
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Using regulatory information to manipulate glycerol metabolism in Saccharomyces cerevisiae
- 2010
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 85:4, s. 1123-1130
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Tidskriftsartikel (refereegranskat)abstract
- Metabolic engineering has emerged as an attractive alternative to random mutagenesis and screening to design cell factories for industrial fermentation processes. The design of metabolic networks has been realized by gene deletions or strong overexpression of heterologous genes. There is an increasing body of evidence that indicates complete inactivation of native genes and high-level activity of heterologous enzymes may be deleterious to the cell. To moderately implement their expression, genes of interest are expressed under the control of promoters with different strengths. Constructing a promoter library is labor-intensive and requires precise quantification of the promoter strength. However, when the mechanisms of pathway regulation are known, it is possible to exploit this information to effect genetic changes efficiently. We report the implementation of this concept to reducing glycerol production during aerobic growth of Saccharomyces cerevisiae. Glycerol is produced to dispose excess cytosolic reduced nicotinamide adenine dinucleotide (NADH), and the regulating step in the pathway is mediated by glycerol 3-phosphate dehydrogenase (encoded by GPD1 and GPD2 genes). We expressed NADH oxidase in S. cerevisiae under the control of the GPD2 promoter to modulate the decrease in cytosolic NADH to the right level where the heterologous enzyme does not compete with oxidative phosphorylation while at the same time, decreasing glycerol production. This metabolic design resulted in substantially decreasing glycerol production and indeed, the excess carbon was redirected to biomass, resulting in a 14% increase in the specific growth rate. We believe that such strategies are more efficient than conventional methods and will find applications in bioprocesses.
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| 7. |
- Jethava, Vinay, 1982, et al.
(författare)
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NETGEM: Network Embedded Temporal GEnerative Model for gene expression data
- 2011
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Ingår i: BMC Bioinformatics. - : Springer Science and Business Media LLC. - 1471-2105. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Temporal analysis of gene expression data has been limited to identifying genes whose expression varies with time and/or correlation between genes that have similar temporal profiles. Often, the methods do not consider the underlying network constraints that connect the genes. It is becoming increasingly evident that interactions change substantially with time. Thus far, there is no systematic method to relate the temporal changes in gene expression to the dynamics of interactions between them. Information on interaction dynamics would open up possibilities for discovering new mechanisms of regulation by providing valuable insight into identifying time-sensitive interactions as well as permit studies on the effect of a genetic perturbation.Results: We present NETGEM, a tractable model rooted in Markov dynamics, for analyzing the dynamics of the interactions between proteins based on the dynamics of the expression changes of the genes that encode them. The model treats the interaction strengths as random variables which are modulated by suitable priors. This approach is necessitated by the extremely small sample size of the datasets, relative to the number of interactions. The model is amenable to a linear time algorithm for efficient inference. Using temporal gene expression data, NETGEM was successful in identifying (i) temporal interactions and determining their strength, (ii) functional categories of the actively interacting partners and (iii) dynamics of interactions in perturbed networks.Conclusions: NETGEM represents an optimal trade-off between model complexity and data requirement. It was able to deduce actively interacting genes and functional categories from temporal gene expression data. It permits inference by incorporating the information available in perturbed networks. Given that the inputs to NETGEM are only the network and the temporal variation of the nodes, this algorithm promises to have widespread applications, beyond biological systems.
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| 8. |
- Petranovic Nielsen, Dina, 1975, et al.
(författare)
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Impact of yeast systems biology on industrial biotechnology
- 2009
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 144:3, s. 204-211
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Tidskriftsartikel (refereegranskat)abstract
- Systems biology is yet an emerging discipline that aims to quantitatively describe and predict the functioning of a biological system. This nascent discipline relies on the recent advances in the analytical technology (such as DNA microarrays, mass spectromety. etc.) to quantify cellular characteristics (such as gene expression, protein and metabolite abundance, etc.) and computational methods to integrate information from these measurements. The model eukaryote, Saccharomyces cerevisiae, has played a pivotal role in the development of many of these analytical and computational methods and consequently is the biological system of choice for testing new hypotheses. The knowledge gained from such studies in S. cerevisiae is proving to be extremely useful in designing metabolism that is targeted to specific industrial applications. As a result, the portfolio of products that are being produced using this yeast is expanding rapidly. We review the recent developments in yeast systems biology and how they relate to industrial biotechnology. (C) 2009 Elsevier B.V. All rights reserved.
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| 9. |
- Petranovic Nielsen, Dina, 1975, et al.
(författare)
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Prospects of yeast systems biology for human health: integrating lipid, protein and energy metabolism
- 2010
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 10:8, s. 1046-1059
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is a widely used model organism for studying cell biology, metabolism, cell cycle and signal transduction. Many regulatory pathways are conserved between this yeast and humans, and it is therefore possible to study pathways that are involved in disease development in a model organism that is easy to manipulate and that allows for detailed molecular studies. Here, we briefly review pathways involved in lipid metabolism and its regulation, the regulatory network of general metabolic regulator Snf1 (and its human homologue AMPK) and the proteostasis network with its link to stress and cell death. All the mentioned pathways can be used as model systems for the study of homologous pathways in human cells and a failure in these pathways is directly linked to several human diseases such as the metabolic syndrome and neurodegeneration. We demonstrate how different yeast pathways are conserved in humans, and we discuss the possibilities of using the systems biology approach to study and compare the pathways of relevance with the objective to generate hypotheses and gain new insights.
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| 10. |
- Vemuri, Goutham, 1976, et al.
(författare)
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Yeast as a Prototype for Systems Biology
- 2009
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Ingår i: Systems Biology and Synthetic Biology. - Hoboken, NJ, USA : John Wiley & Sons, Inc.. - 9780471767787 ; , s. 287-353
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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