| 1. |
- Almquist, Joachim, 1980, et al.
(författare)
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Kinetic models in industrial biotechnology - Improving cell factory performance
- 2014
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 24, s. 38-60
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Tidskriftsartikel (refereegranskat)abstract
- An increasing number of industrial bioprocesses capitalize on living cells by using them as cell factories that convert sugars into chemicals. These processes range from the production of bulk chemicals in yeasts and bacteria to the synthesis of therapeutic proteins in mammalian cell lines. One of the tools in the continuous search for improved performance of such production systems is the development and application of mathematical models. To be of value for industrial biotechnology, mathematical models should be able to assist in the rational design of cell factory properties or in the production processes in which they are utilized. Kinetic models are particularly suitable towards this end because they are capable of representing the complex biochemistry of cells in a more complete way compared to most other types of models. They can, at least in principle, be used to in detail understand, predict, and evaluate the effects of adding, removing, or modifying molecular components of a cell factory and for supporting the design of the bioreactor or fermentation process. However, several challenges still remain before kinetic modeling will reach the degree of maturity required for routine application in industry. Here we review the current status of kinetic cell factory modeling. Emphasis is on modeling methodology concepts, including model network structure, kinetic rate expressions, parameter estimation, optimization methods, identifiability analysis, model reduction, and model validation, but several applications of kinetic models for the improvement of cell factories are also discussed.
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| 2. |
- Aslan, Selcuk, et al.
(författare)
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Wax esters of different compositions produced via engineering of leaf chloroplast metabolism in Nicotiana benthamiana
- 2014
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 25, s. 103-112
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Tidskriftsartikel (refereegranskat)abstract
- In a future bin based economy, renewable sources for lipid compounds at attractive cost are needed for applications where today petrochemical derivatives are dominating. Wax esters and fatty alcohols provide diverse industrial uses, such as in lubricant and surfactant production. In this study, chloroplast metabolism was engineered to divert intermediates from de nova fatty acid biosynthesis to wax ester synthesis. To accomplish this, chloroplast targeted fatty acyl recluctases (EAR) and wax ester synthases (WS) were transiently expressed in Nic"onana benthamiuna loaves. Wax esters of different qualities and quantities were produced providing insights to the properties and interaction of the individual enzymes used. In particular, a phytyl ester synthase was found to be a premium candidate for medium chain wax ester synthesis. Catalytic activities of FAR and WS were also expressed as a fusion protein and determined functionally equivalent to the expression of individual enzymes for wax ester synthesis in chloroplasts. (C) 2014 The Authors. Published by Elsevier Inc. On behalf of International Metabolic Engineering Society.
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| 3. |
- Baebprasert, Wipawee, et al.
(författare)
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Increased H(2) production in the cyanobacterium Synechocystis sp strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway
- 2011
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 13:5, s. 610-616
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Tidskriftsartikel (refereegranskat)abstract
- The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 contains a single bidirectional NiFe-Hox-hydrogenase, which evolves hydrogen under certain environmental conditions. The nitrate assimilation pathway is a potential competing pathway that may reduce the electron flow to the hydrogenase and thereby limit hydrogen production. To improve H(2) production, the nitrate assimilation pathway was disrupted by genetic engineering to redirect the electron flow towards the Hox-hydrogenase. Mutant strains disrupted in either nitrate reductase (Delta narB) or nitrite reductase (Delta nirA) or both nitrate reductase and nitrite reductase (Delta narB:Delta nirA) were constructed and tested for their ability to produce hydrogen. H(2) production and Hox-hydrogenase activities in all the mutant strains were higher than those in wild-type. Highest H(2) production was observed in the Delta narB:Delta nirA strain. Small changes were observed for Hox-hydrogenase enzyme activities and only minor changes in transcript levels of hoxH and hoxY were not correlated with H(2) production. The results suggest that the high rate of H(2) production observed in the Delta narB:Delta nirA strain of the cyanobacterium Synechocystis sp. strain PCC 6803 is the result of redirecting the electron supply from the nitrate assimilation pathway, through genetic engineering, towards the Hox-hydrogenase.
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| 4. |
- Chen, Yun, 1978, et al.
(författare)
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Coupled incremental precursor and co-factor supply improves 3-hydroxypropionic acid production in Saccharomyces cerevisiae
- 2014
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 22, s. 104-109
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Tidskriftsartikel (refereegranskat)abstract
- 3-Hydroxypropionic acid (3-HP) is an attractive platform chemical, which can be used to produce a variety of commodity chemicals, such as acrylic acid and acrylamide. For enabling a sustainable alternative to petrochemicals as the feedstock for these commercially important chemicals, fermentative production of 3-HP is widely investigated and is centered on bacterial systems in most cases. However, bacteria present certain drawbacks for large-scale organic acid production. In this study, we have evaluated the production of 3-HP in the budding yeast Saccharomyces cerevisiae through a route from malonyl-CoA, because this allows performing the fermentation at low pH thus making the overall process cheaper. We have further engineered the host strain by increasing availability of the precursor malonyl-CoA and by coupling the production with increased NADPH supply we were able to substantially improve 3-HP production by five-fold, up to a final titer of 463 mg l(-1). Our work thus led to a demonstration of 3-HP production in yeast via the malonyl-CoA pathway, and this opens for the use of yeast as a cell factory for production of bio-based 3-HP and derived acrylates in the future. (C) 2014 International Metabolic Engineering Society.
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| 5. |
- Chen, Yun, 1978, et al.
(författare)
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Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism
- 2013
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 15:1, s. 48-54
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Tidskriftsartikel (refereegranskat)abstract
- Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible cell factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast cell factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform cell factory can be used to improve the production of alpha-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products.
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| 6. |
- Hong, Kuk-ki, 1976, et al.
(författare)
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Adaptively evolved yeast mutants on galactose show trade-offs in carbon utilization on glucose
- 2013
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 16:1, s. 78-86
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Tidskriftsartikel (refereegranskat)abstract
- Adaptive evolution offers many opportunities in metabolic engineering; however, several constraints still exist as evolutionary trade-offs may impose collateral cost to obtain new traits. The application of adaptive evolution for strains development could be further improved by elucidating the molecular mechanisms. In this study, adaptively evolved yeast mutants with improved galactose utilization ability showed impaired glucose utilization. The molecular genetic basis of this trade-off was investigated using a systems biology approach. Transcriptional and metabolic changes resulting from the improvement of galactose utilization were found maintained during growth on glucose. Moreover, glucose repression related genes showed conserved expression patterns during growth on both sugars. Mutations in the RAS2 gene that were identified as beneficial for galactose utilization in evolved mutants exhibited significant correlation with attenuation of glucose utilization. These results indicate that antagonistic pleiotropy is the dominant mechanism in the observed trade-off, and it is likely realized by changes in glucose signaling.
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| 7. |
- Hou, Jin, 1982, et al.
(författare)
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Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae
- 2012
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 14:2, s. 120-127
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Secl/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and alpha-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases alpha-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae.
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| 8. |
- Kildegaard, Kanchana R., et al.
(författare)
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Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance
- 2014
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 26, s. 57-66
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Tidskriftsartikel (refereegranskat)abstract
- Biologically produced 3-hydroxypropionic acid (3HP) is a potential source for sustainable acrylates and can also find direct use as monomer in the production of biodegradable polymers. For industrial scale production there is a need for robust cell factories tolerant to high concentration of 3HP, preferably at low pH. Through adaptive laboratory evolution we selected S. cerevisiae strains with improved tolerance to 3HP at pH 3.5. Genome sequencing followed by functional analysis identified the causal mutation in SFA1 gene encoding S-(hyclroxymerhyl)glutathione dehydrogenase. Based on our findings, we propose that 3HP toxicity is mediated by 3-hydroxypropionic aldehyde (reuterin ) and that glutathione-dependent reactions are used for reuterin detoxification. The identified molecular response to 3HP and reuterin may well be a general mechanism for handling resistance to organic acid and aldehydes by living cells. (C) 2014 International Metabolic Engineering Society Published by Elsevier Inc. On behalf of International Metabolic Engineering Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/)
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| 9. |
- Lindberg, Pia, et al.
(författare)
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Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism.
- 2010
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 12:1, s. 70-9
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Tidskriftsartikel (refereegranskat)abstract
- The concept of "photosynthetic biofuels" envisions application of a single organism, acting both as photo-catalyst and producer of ready-made fuel. This concept was applied upon genetic engineering of the cyanobacterium Synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from CO(2) and H(2)O. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light-intensity-dependent isoprene synthase expression. Results showed that oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO(2), without a prior requirement for the harvesting, dewatering and processing of the respective biomass.
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| 10. |
- Liu, Lifang, 1979, et al.
(författare)
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Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae
- 2014
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 21, s. 9-16
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Tidskriftsartikel (refereegranskat)abstract
- Due to limitations associated with whole blood for transfusions (antigen compatibility, transmission of infections, supply and storage), the use of cell-free hemoglobin as an oxygen carrier substitute has been in the center of research interest for decades. Human hemoglobin has previously been synthesized in yeast, however the challenge is to balance the expression of the two different globin subunits, as well as the supply of the prosthetic heme required for obtaining the active hemoglobin (alpha(2)beta(2)). In this work we evaluated the expression of different combinations of alpha and beta peptides and combined this with metabolic engineering of the heme biosynthetic pathway. Through evaluation of several different strategies we showed that engineering the biosynthesis pathway can substantially increase the heme level in yeast cells, and this resulted in a significant enhancement of human hemoglobin production. Besides demonstration of improved hemoglobin production our work demonstrates a novel strategy for improving the production of complex proteins, especially multimers with a prosthetic group. Crown Copyright (C) 2013 Published by Elsevier Inc. on behalf of International Metabolic Engineering Society. All rights reserved.
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| 11. |
- Mapelli, Valeria, 1978, et al.
(författare)
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Metabolic and Bioprocess Engineering for Production of selenized yeast with increased content of seleno-methylselenocysteine
- 2011
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 13:3, s. 282-293
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Tidskriftsartikel (refereegranskat)abstract
- Specific Se-metabolites have been recognized as main responsible for beneficial effects of Se-enriched diet and Se-methylselenocysteine (SeMCys) is thought to be among the most effective ones. Here we show that an engineered Saccharomyces cerevisiae strain expressing a codon optimized heterologous selenocysteine-methyltransferase and endowed with high intracellular levels of S-adenosyl-methionine was able to accumulate SeMCys at levels higher than commercial selenized yeasts. A fine tuned carbon- and sulphate-limited fed-batch bioprocess was crucial to achieve good yields of biomass and SeMCys. Through the coupling of metabolic and bioprocess engineering we achieved a ~24-fold increase in SeMCys, compared to certified reference material of selenized yeast. In addition, we investigated the interplay between sulphur and selenium metabolism and the possibility that redox imbalance occurred along with intracellular accumulation of Se. Collectively, our data show how the combination of metabolic and bioprocess engineering can be used for the production of selenized yeast enriched with beneficial Se-metabolites.
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| 12. |
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| 13. |
- Velasco, Sergio, 1980, et al.
(författare)
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Identification of flux control in metabolic networks using non-equilibrium thermodynamics
- 2010
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 12:4, s. 369-377
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Tidskriftsartikel (refereegranskat)abstract
- A method is presented to identify flux controlling reactions in metabolic networks using experimentally determined flux distributions. The method is based on the application of Ziegler's principle for the maximization of entropy production. According to this principle a metabolic network tends to maximize the entropy production rate while satisfying mass balances and maximal rate constraints. Experimental flux data corresponding to four different metabolic states of Saccharomyces cerevisiae were used to identify the corresponding flux controlling reactions. The bottleneck nature of several of the identified reactions was confirmed by earlier studies on over-expression of the identified target genes. The method also explains the failure of all the previous trials of increasing the glycolysis rate by direct over-expression of several glycolytic enzymes. These findings point to a wider use of the method for identification of novel targets for metabolic engineering of microorganisms used for sustainable production of fuels and chemicals.
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| 14. |
- Skorupa Parachin, Nadia, et al.
(författare)
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Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae.
- 2011
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176. ; 13, s. 508-517
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.
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| 15. |
- Willquist, Karin, et al.
(författare)
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Lactate formation in Caldicellulosiruptor saccharolyticus is regulated by the energy carriers pyrophosphate and ATP
- 2010
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176. ; 12, s. 282-290
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Tidskriftsartikel (refereegranskat)abstract
- Caldicellulosiruptor saccharolyticus displays superior H(2) yields on a wide range of carbon sources provided that lactate formation is avoided. Nevertheless, a low lactate flux is initiated as the growth rate declined in the transition to the stationary phase, which coincides with a drastic decrease in the glucose consumption and acetate production fluxes. In addition, the decrease in growth rate was accompanied by a sudden increase and then decrease in NADH levels. The V'(MAX) of the lactate dehydrogenase (LDH) doubled when the cells entered the stationary phase. Kinetic analysis revealed that at the metabolic level LDH activity is regulated through (i) competitive inhibition by pyrophosphate (PPi, k(i)=1.7mM) and NAD (k(i)=0.43mM) and (ii) allosteric activation by FBP (300%), ATP (160%) and ADP (140%). From these data a MWC-based model was derived. Simulations with this model could explain the observed lactate shift by displaying how the sensitivity of LDH activity to NADH/NAD ratio varied with different PP(i) concentrations. Moreover, the activation of LDH by ATP indicates that C. saccharolyticus uses LDH as a means to adjusts its flux of ATP and NADH production. To our knowledge, this is the first time PPi is observed as an effector of LDH.
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