| 1. |
- Bengtsson, Oskar, et al.
(författare)
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Identification of common traits in improved xylose-growing Saccharomyces cerevisiae for inverse metabolic engineering.
- 2008
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 25:11, s. 835-847
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Tidskriftsartikel (refereegranskat)abstract
- Four recombinant Saccharomyces cerevisiae strains with enhanced xylose growth (TMB3400, C1, C5 and BH42) were compared with two control strains (TMB3399, TMB3001) through genome-wide transcription analysis in order to identify novel targets for inverse metabolic engineering. A subset of 13 genes with changed expression levels in all improved strains was selected for further analysis. Thirteen validation strains and two reference strains were constructed to investigate the effect of overexpressing or deleting these genes in xylose-utilizing S. cerevisiae. Improved aerobic growth rates on xylose were observed in five cases. The strains overexpressing SOL3 and TAL1 grew 19% and 24% faster than their reference strain, and the strains carrying deletions of YLR042C, MNI1 or RPA49 grew 173%, 62% and 90% faster than their reference strain.
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| 2. |
- Gárdonyi, Márk, et al.
(författare)
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Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae
- 2003
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 7:82, s. 818-824
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Tidskriftsartikel (refereegranskat)abstract
- Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization. We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, C, for the xylose transport was calculated from the response and elasticity coefficients. The value of C for both strains was found to be < 0.1 at extracellular xylose concentrations > 7.5 g L-1. However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L-1, while C stayed below 0.2 for strain TMB3001 irrespective of xylose concentration. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 818-824, 2003.
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| 3. |
- Hahn-Hägerdal, Bärbel, et al.
(författare)
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Metabolic engineering for pentose utilization in Saccharomyces cerevisiae
- 2007
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Ingår i: Advances in Biochemical Engineering/Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. - 9783540736509 ; 108, s. 147-177
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The introduction of pentose utilization pathways in baker's yeast Saccharomyces cerevisiae is summarized together with metabolic engineering strategies to improve ethanolic pentose fermentation. Bacterial and fungal xylose and arabinose pathways have been expressed in S. cerevisiae but do not generally convey significant ethanolic fermentation traits to this yeast. A large number of rational metabolic engineering strategies directed among others toward sugar transport, initial pentose conversion, the pentose phosphate pathway, and the cellular redox metabolism have been exploited. The directed metabolic engineering approach has often been combined with random approaches including adaptation, mutagenesis, and hybridization. The knowledge gained about pentose fermentation in S. cerevisiae is primarily limited to genetically and physiologically well-characterized laboratory strains. The translation of this knowledge to strains performing in an industrial context is discussed.
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| 4. |
- Jeppsson, Marie, et al.
(författare)
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Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae
- 2003
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Ingår i: FEMS Yeast Research. - 1567-1364. ; 3:2, s. 167-175
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (xylose reductase and xylitol dehydrogenase, respectively) and the endogenous XKS1(xylulokinase), can convert xylose to ethanol. About 30% of the consumed xylose, however, is excreted as xylitol. Enhanced ethanol yield has previously been achieved by disrupting the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase, but at the expense of the xylose consumption. This is probably the result of reduced NADPH-mediated xylose reduction. In the present study, we increased the xylose reductase (XR) activity 4–19 times in both TMB3001 and the ZWF1-disrupted strain TMB3255. The xylose consumption rate increased by 70% in TMB3001 under oxygen-limited conditions. In the ZWF1-disrupted background, the increase in XR activity fully restored the xylose consumption rate. Maximal specific growth rates on glucose were lower in the ZWF1-disrupted strains, and the increased XR activity also negatively affected the growth rate in these strains. Addition of methionine resulted in 70% and 50% enhanced maximal specific growth rates for TMB3255 (zwf1Δ) and TMB3261 (PGK1-XYL1, zwf1Δ), respectively. Enhanced XR activity did not have any negative effect on the maximal specific growth rate in the control strain. Enhanced glycerol yields were observed in the high-XR-activity strains. These are suggested to result from the observed reductase activity of the purified XR for dihydroxyacetone phosphate.
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| 5. |
- Jeppsson, Marie, et al.
(författare)
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Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose.
- 2002
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Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 68:4, s. 1604-1609
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Tidskriftsartikel (refereegranskat)abstract
- In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.
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| 6. |
- Jeppsson, Marie, et al.
(författare)
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The expression of a Pichia stipitis xylose reductase mutant with higher K-M for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae
- 2006
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 93:4, s. 665-673
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Tidskriftsartikel (refereegranskat)abstract
- Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K-M for NADPH (Kostrzynska et al., 1998: FEMS Microbiol Lett 159:107-112) was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source. (c) 2005 Wiley Periodicals, Inc.
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| 7. |
- Jeppsson, Marie, et al.
(författare)
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The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains.
- 2003
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 20:15, s. 1263-1272
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Tidskriftsartikel (refereegranskat)abstract
- Disruption of the ZWF1 gene encoding glucose-6-phosphate dehydrogenase (G6PDH) has been shown to reduce the xylitol yield and the xylose consumption in the xylose-utilizing recombinant Saccharomyces cerevisiae strain TMB3255. In the present investigation we have studied the influence of different production levels of G6PDH on xylose fermentation. We used a synthetic promoter library and the copper-regulated CUP1 promoter to generate G6PDH-activities between 0% and 179% of the wild-type level. G6PDH-activities of 1% and 6% of the wild-type level resulted in 2.8- and 5.1-fold increase in specific xylose consumption, respectively, compared with the ZWF1-disrupted strain. Both strains exhibited decreased xylitol yields (0.13 and 0.19 g/g xylose) and enhanced ethanol yields (0.36 and 0.34 g/g xylose) compared with the control strain TMB3001 (0.29 g xylitol/g xylose, 0.31 g ethanol/g xylose). Cytoplasmic transhydrogenase (TH) from Azotobacter vinelandii has previously been shown to transfer NADPH and NAD+ into NADP+ and NADH, and TH-overproduction resulted in lower xylitol yield and enhanced glycerol yield during xylose utilization. Strains with low G6PDH-activity grew slower in a lignocellulose hydrolysate than the strain with wild-type G6PDH-activity, which suggested that the availability of intracellular NADPH correlated with tolerance towards lignocellulose-derived inhibitors. Low G6PDH-activity strains were also more sensitive to H2O2 than the control strain TMB3001. Copyright © 2003 John Wiley & Sons, Ltd.
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| 8. |
- Sonderegger, M, et al.
(författare)
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Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains
- 2004
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 87:1, s. 90-98
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Tidskriftsartikel (refereegranskat)abstract
- Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate-containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate. (C) 2004 Wiley Periodicals, Inc.
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| 9. |
- Träff, Karin, et al.
(författare)
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Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae
- 2004
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Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 21:2, s. 141-150
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Tidskriftsartikel (refereegranskat)abstract
- Introduction of the xylose pathway from Pichia stipitis into Saccharomyces cerevisiae enables xylose utilization in recombinant S. cerevisiae. However, xylitol is a major by-product. An endogenous aldo-keto reductase, encoded by the GRE3 gene, was expressed at different levels in recombinant S. cerevisiae strains to investigate its effect on xylose utilization. In a recombinant S. cerevisiae strain producing only xylitoll dehydrogenase (XDH) from P. stipitis and an extra copy of the endogenous xylulokinase (XK), ethanol formation from xylose was mediated by Gre3p, capable of reducing xylose to xylitol. When the GRE3 gene was overexpressed in this strain, the xylose consumption and ethanol formation increased by 29% and 116%, respectively. When the GRE3 gene was deleted in the recombinant xylose-fermenting S. cerevisiae strain TMB3001 (which possesses xylose reductase and XDH from P. stipitis, and an extra copy of endogenous XK), the xylitol yield decreased by 49% and the ethanol yield increased by 19% in anaerobic continuous culture with a glucose/xylose mixture. Biomass was reduced by 31% in strains where GRE3 was deleted, suggesting that fine-tuning of GRE3 expression is the preferred choice rather than deletion. Copyright (C) 2003 John Wiley Sons, Ltd.
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| 10. |
- Abdelaziz, Omar Y., et al.
(författare)
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Biological valorization of low molecular weight lignin
- 2016
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Ingår i: Biotechnology Advances. - : Elsevier BV. - 0734-9750. ; 34:8, s. 1318-1346
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Forskningsöversikt (refereegranskat)abstract
- Lignin is a major component of lignocellulosic biomass and as such, it is processed in enormous amounts in the pulp and paper industry worldwide. In such industry it mainly serves the purpose of a fuel to provide process steam and electricity, and to a minor extent to provide low grade heat for external purposes. Also from other biorefinery concepts, including 2nd generation ethanol, increasing amounts of lignin will be generated. Other uses for lignin – apart from fuel production – are of increasing interest not least in these new biorefinery concepts. These new uses can broadly be divided into application of the polymer as such, native or modified, or the use of lignin as a feedstock for the production of chemicals. The present review focuses on the latter and in particular the advances in the biological routes for chemicals production from lignin. Such a biological route will likely involve an initial depolymerization, which is followed by biological conversion of the obtained smaller lignin fragments. The conversion can be either a short catalytic conversion into desired chemicals, or a longer metabolic conversion. In this review, we give a brief summary of sources of lignin, methods of depolymerization, biological pathways for conversion of the lignin monomers and the analytical tools necessary for characterizing and evaluating key lignin attributes.
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