| 1. |
- Almeida, Joao, et al.
(author)
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Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate.
- 2009
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In: Bioresource Technology. - : Elsevier BV. - 1873-2976 .- 0960-8524. ; 100, s. 3674-3677
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Journal article (peer-reviewed)abstract
- A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae strains in barley straw, spruce and wheat straw hydrolysate. The assay demonstrated significant differences in inhibitor tolerance among the strains. In addition, growth inhibition by the three hydrolysates differed so that wheat hydrolysate supported growth up to 70%, while barley hydrolysate only supported growth up to 50%, with dilute-acid spruce hydrolysate taking an intermediate position.
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| 2. |
- Garcia Sanchez, Rosa, et al.
(author)
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Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering
- 2010
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In: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 3
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Journal article (peer-reviewed)abstract
- BACKGROUND: Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced. RESULTS: Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways. CONCLUSION: To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.
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| 3. |
- Hahn-Hägerdal, Bärbel, et al.
(author)
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Metabolic engineering for pentose utilization in Saccharomyces cerevisiae
- 2007
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In: Advances in Biochemical Engineering/Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. - 9783540736509 ; 108, s. 147-177
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Book chapter (other academic/artistic)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. |
- Hahn-Hägerdal, Bärbel, et al.
(author)
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Role of cultivation media in the development of yeast strains for large scale industrial use
- 2005
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 4:Art. No. 31
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Research review (peer-reviewed)abstract
- The composition of cultivation media in relation to strain development for industrial application is reviewed. Heterologous protein production and pentose utilization by Saccharomyces cerevisiae are used to illustrate the influence of media composition at different stages of strain construction and strain development. The effects of complex, defined and industrial media are compared. Auxotrophic strains and strain stability are discussed. Media for heterologous protein production and for bulk bio-commodity production are summarized.
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| 5. |
- Hahn-Hägerdal, Bärbel, et al.
(author)
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Towards industrial pentose-fermenting yeast strains
- 2007
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 74:5, s. 937-953
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Research review (peer-reviewed)abstract
- Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.
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| 6. |
- Hambraeus, Gustav, et al.
(author)
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A 5' stem-loop and ribosome binding but not translation are important for the stability of Bacillus subtilis aprE leader mRNA.
- 2002
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In: Microbiology. - 1465-2080. ; 148:Pt 6, s. 1795-1803
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Journal article (peer-reviewed)abstract
- The Bacillus subtilis aprE leader is a determinant of extreme mRNA stability. The authors examined what properties of the aprE leader confer stability on an mRNA. The secondary structure of the aprE leader mRNA was analysed in vitro and in vivo, and mutations were introduced into different domains of an aprE leader-lacZ fusion. The half-lives of the corresponding transcripts were determined and beta-galactosidase activities were measured. Removal of a stem-loop structure at the 5' end or diminishing the strength of the RBS reduced the half-lives from more than 25 min to about 5 min. Interfering with translation by abolishing the start codon or creating an early stop codon had no or little effect on mRNA stability. The authors conclude that a 5' stem-loop and binding of ribosomes are necessary for the stability of aprE leader mRNA. The present results, together with a number of other data, suggest that translation of a B. subtilis mRNA is generally not important for its stability; the situation seems different in Escherichia coli. It is further concluded that the calculated strength of a B. subtilis RBS cannot be used to predict the stability of the corresponding transcript.
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| 7. |
- Karhumaa, Kaisa, et al.
(author)
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Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains
- 2006
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 5
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Journal article (peer-reviewed)abstract
- Background: Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. Results: We describe the engineering of laboratory and industrial S. cerevisiae strains to co-ferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. Conclusion: Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.
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| 8. |
- Karhumaa, Kaisa, et al.
(author)
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Comparison of xylose fermentation by Saccharomyces cerevisiae strains carrying either xylose reductase and xylitol dehydrogenase or xylose isomerase
- 2007
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 6:1
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Journal article (peer-reviewed)abstract
- Background: Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase ( XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate. Results: In defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected. Conclusion: Despite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed.
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| 9. |
- Karhumaa, Kaisa
(author)
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Engineering xylose and arabinose metabolism in recombinant Saccharomyces cerevisiae
- 2006
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Doctoral thesis (other academic/artistic)abstract
- Utilization of all sugars in lignocellulose hydrolysates is a prerequisite for economically feasible bioethanol production. The yeast commonly used for industrial ethanol production, Saccharomyces cerevisiae, is naturally unable to utilize pentose sugars xylose and arabinose, which constitute a large fraction of many lignocellulosic materials. Xylose utilization by S. cerevisiae can be achieved by heterologous expression of a xylose utilization pathway, consisting either of xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK), or alternatively, of xylose isomerase (XI) and XK. Xylitol formed by XR is a major by-product in xylose fermentation when using the XR-XDH pathway. In this thesis, high-level expression of both XR and XDH was shown to decrease xylitol formation. The influence of other genetic modifications was also evaluated. It was shown that the overexpression of the non-oxidative pentose phosphate pathway (PPP) genes enables efficient growth on xylose and xylose fermentation, provided that the initial xylose pathway is expressed at a high level. When comparing the two xylose utilization pathways, higher ethanol productivity was achieved using the XR-XDH pathway, whereas higher ethanol yield was achieved with the XI pathway. The industrial xylose-fermenting S. cerevisiae strain TMB 3400, which has been previously generated by mutagenesis and selection, was tested for fermentation of lignocellulose hydrolysate. TMB 3400 displayed significantly better fermentation performance compared to the laboratory strains tested, highlighting the need for robust industrial strains in lignocellulose fermentation. TMB 3400 was also characterized by proteome analysis using difference in-gel 2-D electrophoresis. Consistently with the results obtained in other studies, increased activities of XR, XDH and a PPP enzyme TKL were found. The bacterial arabinose utilization pathway was introduced into TMB 3400, which resulted in the novel glucose, xylose and arabinose co-fermenting strain TMB 3063, with ethanol, xylitol and arabitol as the main fermentation products.
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| 10. |
- Karhumaa, Kaisa, et al.
(author)
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High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae
- 2007
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In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 73:5, s. 1039-1046
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Journal article (peer-reviewed)abstract
- Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.
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