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1.
  • Gárdonyi, Márk, et al. (författare)
  • Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae
  • 2003
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 7:82, s. 818-824
  • 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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2.
  • Hahn-Hägerdal, Bärbel, et al. (författare)
  • Metabolic engineering for pentose utilization in Saccharomyces cerevisiae
  • 2007
  • Ingår i: Advances in Biochemical Engineering/Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. - 9783540736509 ; 108, s. 147-177
  • 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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3.
  • Jeppsson, Marie, et al. (författare)
  • Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae
  • 2003
  • Ingår i: FEMS Yeast Research. - 1567-1364. ; 3:2, s. 167-175
  • 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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4.
  • Jeppsson, Marie, et al. (författare)
  • Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose.
  • 2002
  • Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 68:4, s. 1604-1609
  • 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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5.
  • Jeppsson, Marie, et al. (författare)
  • 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
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 93:4, s. 665-673
  • 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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6.
  • Jeppsson, Marie, et al. (författare)
  • The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains.
  • 2003
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 20:15, s. 1263-1272
  • 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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7.
  • Sonderegger, M, et al. (författare)
  • Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains
  • 2004
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 87:1, s. 90-98
  • 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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8.
  • Träff, Karin, et al. (författare)
  • Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae
  • 2004
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 21:2, s. 141-150
  • 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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9.
  • Almeida, Joao, et al. (författare)
  • Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction.
  • 2009
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84, s. 751-761
  • Tidskriftsartikel (refereegranskat)abstract
    • Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD(+)-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD(+) deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.
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10.
  • Almeida, Joao, et al. (författare)
  • Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae
  • 2007
  • Ingår i: Journal of Chemical Technology and Biotechnology. - : Wiley. - 0268-2575 .- 1097-4660. ; 82:4, s. 340-349
  • Forskningsöversikt (refereegranskat)abstract
    • During hydrolysis of lignocellulosic biomass, monomeric sugars and a broad range of inhibitory compounds are formed and released. These inhibitors, which can be organized around three main groups, furans, weak acids and phenolics, reduce ethanol yield and productivity by affecting the microorganism performance during the fermentation step. Among the microorganisms that have been evaluated for lignocellulosic hydrolysate ethanol fermentation, the yeast Saccharomyces cerevisiae appears to be the least sensitive. In order to overcome the effect of inhibitors, strategies that include improvement of natural tolerance of microorganism and use of fermentation control strategies have been developed. An overview of the origin, effects and mechanisms of action of known inhibitors on S. cerevisiae is given. Fermentation control strategies as well as metabolic, genetic and evolutionary engineering strategies to obtain S. cerevisiae strains with improved tolerance are discussed.
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11.
  • Almeida, Joao, et al. (författare)
  • Metabolic effects of furaldehydes and impacts on biotechnological processes.
  • 2009
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 82:4, s. 625-638
  • Forskningsöversikt (refereegranskat)abstract
    • There is a growing awareness that lignocellulose will be a major raw material for production of both fuel and chemicals in the coming decades-most likely through various fermentation routes. Considerable attention has been given to the problem of finding efficient means of separating the major constituents in lignocellulose (i.e., lignin, hemicellulose, and cellulose) and to efficiently hydrolyze the carbohydrate parts into sugars. In these processes, by-products will inevitably form to some extent, and these will have to be dealt with in the ensuing microbial processes. One group of compounds in this category is the furaldehydes. 2-Furaldehyde (furfural) and substituted 2-furaldehydes-most importantly 5-hydroxymethyl-2-furaldehyde-are the dominant inhibitory compounds found in lignocellulosic hydrolyzates. The furaldehydes are known to have biological effects and act as inhibitors in fermentation processes. The effects of these compounds will therefore have to be considered in the design of biotechnological processes using lignocellulose. In this short review, we take a look at known metabolic effects, as well as strategies to overcome problems in biotechnological applications caused by furaldehydes.
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12.
  • Almeida, Joao, et al. (författare)
  • NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae.
  • 2008
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 78:6, s. 939-945
  • Tidskriftsartikel (refereegranskat)abstract
    • Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.
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13.
  • Almeida, Joao, et al. (författare)
  • Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)
  • 2008
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Pichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xyloseconsuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21. Results: In this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher in vitro HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both in vitro and in vivo. Ps-XR overexpression increases the in vivo HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme. Conclusion: We demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.
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14.
  • Almeida, Joao, et al. (författare)
  • Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate.
  • 2009
  • Ingår i: Bioresource Technology. - : Elsevier BV. - 1873-2976 .- 0960-8524. ; 100, s. 3674-3677
  • Tidskriftsartikel (refereegranskat)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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15.
  • Almeida, Joao, et al. (författare)
  • Stress-related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae.
  • 2011
  • Ingår i: Biotechnology Journal. - : Wiley. - 1860-6768. ; 6, s. 286-299
  • Forskningsöversikt (refereegranskat)abstract
    • Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial process conditions is the subject of this review. First, fermentation challenges originating from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence of contaminating microbiota.
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16.
  • Bengtsson, Oskar, et al. (författare)
  • Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae
  • 2009
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 2
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for xylose utilization. The availability of NAD+ for XDH is limited during anaerobic xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference. Results: XR variants were evaluated in S. cerevisiae strains with the following genetic modifications: overexpressed native P. stipitis XDH, overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deleted GRE3 gene encoding an NADPH dependent aldose reductase. All overexpressed genes were chromosomally integrated to ensure stable expression. Crude extracts of four different strains overexpressing genes encoding native P. stipitis XR, K270M and K270R mutants, as well as Candida parapsilosis XR, were enzymatically characterized. The physiological effects of the mutations were investigated in anaerobic xylose fermentation. The strain overexpressing P. stipitis XR with the K270R mutation gave an ethanol yield of 0.39 g (g consumed sugars)(-1), a xylitol yield of 0.05 g (g consumed xylose)(-1) and a xylose consumption rate of 0.28 g (g biomass)(-1) h(-1) in continuous fermentation at a dilution rate of 0.12 h(-1), with 10 g l(-1) glucose and 10 g l(-1) xylose as carbon sources. Conclusion: The cofactor preference of P. stipitis XR was altered by site-directed mutagenesis. When the K270R XR was combined with a metabolic engineering strategy that ensures high xylose utilization capabilities, a recombinant S. cerevisiae strain was created that provides a unique combination of high xylose consumption rate, high ethanol yield and low xylitol yield during ethanolic xylose fermentation.
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17.
  • Bergdahl, Basti, et al. (författare)
  • Engineering yeast hexokinase 2 for improved tolerance toward xylose-induced inactivation.
  • 2013
  • Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:9
  • Tidskriftsartikel (refereegranskat)abstract
    • Hexokinase 2 (Hxk2p) from Saccharomyces cerevisiae is a bi-functional enzyme being both a catalyst and an important regulator in the glucose repression signal. In the presence of xylose Hxk2p is irreversibly inactivated through an autophosphorylation mechanism, affecting all functions. Consequently, the regulation of genes involved in sugar transport and fermentative metabolism is impaired. The aim of the study was to obtain new Hxk2p-variants, immune to the autophosphorylation, which potentially can restore the repressive capability closer to its nominal level. In this study we constructed the first condensed, rationally designed combinatorial library targeting the active-site in Hxk2p. We combined protein engineering and genetic engineering for efficient screening and identified a variant with Phe159 changed to tyrosine. This variant had 64% higher catalytic activity in the presence of xylose compared to the wild-type and is expected to be a key component for increasing the productivity of recombinant xylose-fermenting strains for bioethanol production from lignocellulosic feedstocks.
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18.
  • Bergdahl, Basti, et al. (författare)
  • Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae
  • 2014
  • Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 14:28
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Efficient utilization of both glucose and xylose is necessary for a competitive ethanol production from lignocellulosic materials. Although many advances have been made in the development of xylose-fermenting strains of Saccharomyces cerevisiae, the productivity remains much lower compared to glucose. Previous transcriptional analyses of recombinant xylose-fermenting strains have mainly focused on central carbon metabolism. Very little attention has been given to other fundamental cellular processes such as the folding of proteins. Analysis of previously measured transcript levels in a recombinant XR/XDH-strain showed a wide down-regulation of genes targeted by the unfolded protein response during xylose fermentation. Under anaerobic conditions the folding of proteins is directly connected with fumarate metabolism and requires two essential enzymes: FADH2-dependent fumarate reductase (FR) and Ero1p. In this study we tested whether these enzymes impair the protein folding process causing the very slow growth of recombinant yeast strains on xylose under anaerobic conditions. Results: Four strains over-expressing the cytosolic (FRD1) or mitochondrial (OSM1) FR genes and ERO1 in different combinations were constructed. The growth and fermentation performance was evaluated in defined medium as well as in a complex medium containing glucose and xylose. Over-expression of FRD1, alone or in combination with ERO1, did not have any significant effect on xylose fermentation in any medium used. Over-expression of OSM1, on the other hand, led to a diversion of carbon from glycerol to acetate and a decrease in growth rate by 39% in defined medium and by 25% in complex medium. Combined over-expression of OSM1 and ERO1 led to the same diversion of carbon from glycerol to acetate and had a stronger detrimental effect on the growth in complex medium. Conclusions: Increasing the activities of the FR enzymes and Ero1p is not sufficient to increase the anaerobic growth on xylose. So additional components of the protein folding mechanism that were identified in transcription analysis of UPR related genes may also be limiting. This includes i) the transcription factor encoded by HAC1 ii) the activity of Pdi1p and iii) the requirement of free FAD during anaerobic growth.
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19.
  • Bettiga, Maurizio, et al. (författare)
  • Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway
  • 2009
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 8
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation. Results: A new recombinant S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized. Conclusion: Pentose fermenting recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar.
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20.
  • Bettiga, Maurizio, et al. (författare)
  • Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains
  • 2008
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose. Results: The specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells)-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/ xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)-1 h-1 compared with 0.01 g (g cells)-1 h-1 for the xylose reductase/ xylitol dehydrogenase strain and the xylose isomerase strain, respectively. Conclusion: The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.
  •  
21.
  • Bettiga, Maurizio, et al. (författare)
  • Metabolic engineering in yeast
  • 2009
  • Ingår i: The Metabolic Pathway Engineering Handbook : Fundamentals. - 9781439802960 ; , s. 1-46
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)
  •  
22.
  • Blomqvist, Johanna, et al. (författare)
  • Physiological requirements for growth and competitiveness of Dekkera bruxellensis under oxygen-limited or anaerobic conditions
  • 2012
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 29:7, s. 265-274
  • Tidskriftsartikel (refereegranskat)abstract
    • The effect of glucose and oxygen limitation on the growth and fermentation performances of Dekkera bruxellensis was investigated in order to understand which factors favour its propagation in ethanol or wine plants. Although D. bruxellensis has been described as a facultative anaerobe, no growth was observed in mineral medium under complete anaerobiosis while growth was retarded under severe oxygen limitation. In a continuous culture with no gas inflow, glucose was not completely consumed, most probably due to oxygen limitation. When an air/nitrogen mixture (O-2-content ca. 5%) was sparged to the culture, growth became glucose-limited. In co-cultivations with Saccharomyces cerevisiae, ethanol yields/g consumed sugar were not affected by the co-cultures as compared to the pure cultures. However, different population responses were observed in both systems. In oxygen-limited cultivation, glucose was depleted within 24 h after challenging with S. cerevisiae and both yeast populations were maintained at a stable level. In contrast, the S. cerevisiae population constantly decreased to about 1% of its initial cell number in the sparged glucose-limited fermentation, whereas the D. bruxellensis population remained constant. To identify the requirements of D. bruxellensis for anaerobic growth, the yeast was cultivated in several nitrogen sources and with the addition of amino acids. Yeast extract and most of the supplied amino acids supported anaerobic growth, which points towards a higher nutrient demand for D. bruxellensis compared to S. cerevisiae in anaerobic conditions. Copyright (c) 2012 John Wiley & Sons, Ltd.
  •  
23.
  • Botes, AL, et al. (författare)
  • Screening of yeast species for the stereo-selective reduction of bicyclo[2.2.2]octane-2,6-dione
  • 2002
  • Ingår i: Journal of the Chemical Society - Perkin Transactions 1. - : Royal Society of Chemistry (RSC). - 1472-7781 .- 1364-5463. ; :8, s. 1111-1114
  • Tidskriftsartikel (refereegranskat)abstract
    • Yeast strains from more than 31 different genera were screened for the enantioselective reduction of bicyclo[2.2.2]-octane-2,6-dione (1). Reducing activity was found in 80% of the screened yeasts. Bicyclo[2.2.2]octane-2,6-dione was enantioselectively reduced (> 98% ee) to (1R, 4S, 6S)-6-hydroxybicyclo[2.2.2]octane-2-one (-)-2 by 69% of the strains. Enantioselective reduction of the diketone to (1S, 4R, 6S)-6-hydroxybicyclo[2.2.2]octane-2-one ((+)-3, >98% ee) as a major product is reported for the first time. Candida tropicalis UOFS Y-0534 and Candida wickerhamii UOFS Y-0652 displayed this unusual diastereoselectivity.
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24.
  • Cadete, Raquel M., et al. (författare)
  • Exploring xylose metabolism in Spathaspora species : XYL1.2 from Spathaspora passalidarum as the key for efficient anaerobic xylose fermentation in metabolic engineered Saccharomyces cerevisiae
  • 2016
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 9:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: The production of ethanol and other fuels and chemicals from lignocellulosic materials is dependent of efficient xylose conversion. Xylose fermentation capacity in yeasts is usually linked to xylose reductase (XR) accepting NADH as cofactor. The XR from Scheffersomyces stipitis, which is able to use NADH as cofactor but still prefers NADPH, has been used to generate recombinant xylose-fermenting Saccharomyces cerevisiae. Novel xylose-fermenting yeasts species, as those from the Spathaspora clade, have been described and are potential sources of novel genes to improve xylose fermentation in S. cerevisiae. Results: Xylose fermentation by six strains from different Spathaspora species isolated in Brazil, plus the Sp. passalidarum type strain (CBS 10155T), was characterized under two oxygen-limited conditions. The best xylose-fermenting strains belong to the Sp. passalidarum species, and their highest ethanol titers, yields, and productivities were correlated to higher XR activity with NADH than with NADPH. Among the different Spathaspora species, Sp. passalidarum appears to be the sole harboring two XYL1 genes: XYL1.1, similar to the XYL1 found in other Spathaspora and yeast species and XYL1.2, with relatively higher expression level. XYL1.1p and XYL1.2p from Sp. passalidarum were expressed in S. cerevisiae TMB 3044 and XYL1.1p was confirmed to be strictly NADPH-dependent, while XYL1.2p to use both NADPH and NADH, with higher activity with the later. Recombinant S. cerevisiae strains expressing XYL1.1p did not show anaerobic growth in xylose medium. Under anaerobic xylose fermentation, S. cerevisiae TMB 3504, which expresses XYL1.2p from Sp. passalidarum, revealed significant higher ethanol yield and productivity than S. cerevisiae TMB 3422, which harbors XYL1p N272D from Sc. stipitis in the same isogenic background (0.40 vs 0.34 g g CDW -1 and 0.33 vs 0.18 g g CDW -1 h-1, respectively). Conclusion: This work explored a new clade of xylose-fermenting yeasts (Spathaspora species) towards the engineering of S. cerevisiae for improved xylose fermentation. The new S. cerevisiae TMB 3504 displays higher XR activity with NADH than with NADPH, with consequent improved ethanol yield and productivity and low xylitol production. This meaningful advance in anaerobic xylose fermentation by recombinant S. cerevisiae (using the XR/XDH pathway) paves the way for the development of novel industrial pentose-fermenting strains.
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25.
  • Carlquist, Magnus, et al. (författare)
  • Flavonoids as inhibitors of human carbonyl reductase 1
  • 2008
  • Ingår i: Chemico-Biological Interactions. - : Elsevier BV. - 1872-7786 .- 0009-2797. ; 174:2, s. 98-108
  • Tidskriftsartikel (refereegranskat)abstract
    • Human carbonyl reductase 1 (CBR1), that is one of the enzymes responsible for the reduced efficiency of treatments by the antineoplastic agents anthracyclines, was functionally expressed in Saccharomyces cerevisiae. CBR1 was purified and kinetically characterised using daunorubicin as substrate. CBR1-catalysed reduction of daunorubicin followed an apparent Michaelis-Menten kinetics with K-M = 85.2 +/- 26.7 mu M and V-max =3490 +/- 220 mu mol/(min g protein). The type of inhibition for the flavonoid compound rutin was determined by studying initial reaction rates in the presence of rutin. The inhibition kinetics was found to follow an apparent mixed inhibition with K-ic = 1.8 +/- 1.2 mu M and K-iu = 2.8 +/- 1.6 mu M. IC50-values were also determined for a set of flavonoids in order to identify essential structure for inhibition activity. Computational docking experiments of the four best inhibitors to the catalytic site of CBR1 showed that the flavonoid skeleton structure was the binding part of the molecule. The presence of a sugar moiety in I and 2, or a sugar mimicking part in 9. directed the orientation of the flavonoid so that the sugars were pointing outwards, giving rise to a stabilising effect to the binding. Finally, additional binding epitopes that interacted with various parts of the flavonoid ligand were identified and could potentially be targeted for further improvement of inhibition activity. These included; hydrogen-binding sites surrounding Ser139 and Cys226, Met234 and Tyr193 or Trp229; aromatic-aromatic interaction with Tyr193, Trp229 or NADPH; van der Waals interactions with IIe140. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
  •  
26.
  • Carlquist, Magnus, et al. (författare)
  • Genetically engineered Saccharomyces cerevisiae for kinetic resolution of racemic bicyclo[3.3.1]nonane-2,6-dione
  • 2008
  • Ingår i: Tetrahedron: Asymmetry. - : Elsevier BV. - 0957-4166. ; 19:19, s. 2293-2295
  • Tidskriftsartikel (refereegranskat)abstract
    • Whole cells of the genetically engineered Saccharomyces cerevisiae strain TMB4100 (1% PGI, YMR226c) were Used as the biocatalyst for the kinetic resolution of racemic bicyclo[3.3.1]nonane-2,6-dione rac-1. The yeast's phosphoglucose isomerase activity was decreased, and the short-chain dehydrogenase/reductase encoded by YMR226c was overexpressed. This reduced the demand for the glucose to regenerate NADPH, while at the same time the reaction rate and selectivity towards (-)-1 became higher. The demand for yeast biomass also decreased, facilitating down-stream processing, which is of considerable importance oil a large scale. With 15 g dry weight/L of the genetically engineered yeast TMB4100 (1% PGI, YMR226c), 40 g/L rac-1 was kinetically resolved within 24 h producing pure (+)-1 with all enantiomeric excess (ee) of 100% after 75% conversion. This corresponds to a biochemical selectivity constant of E = 10.3 +/- 2.2. Thus, compared with conventional methods which use commercial baker's yeast as a biocatalyst, the reaction system was significantly improved, and Would be superior in a large-scale process. (C) 2008 Elsevier Ltd. All rights reserved.
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27.
  • Carlquist, Magnus, et al. (författare)
  • Kinetic resolution of racemic 5,6-epoxy-bicyclo[2.2.1]heptane-2-one using genetically engineered Saccharomyces cerevisiae
  • 2009
  • Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 58:2, s. 98-102
  • Tidskriftsartikel (refereegranskat)abstract
    • (+)-5,6-Epoxy-bicyclo[2.2.1]heptane-2-one, (+)-1, and endo-(−)-5,6-epoxy-bicyclo[2.2.1]heptane-2-ol, endo-(−)-2, were obtained by kinetic resolution of rac-1 by asymmetric bioreduction catalyzed by whole cells of a genetically engineered Saccharomyces cerevisiae yeast strain. The strain, TMB4100, had 1% phosphoglucose isomerase (PGI) activity and overexpressed a specific short-chain dehydrogenase, encoded by the gene YMR226c. The whole cell biocatalystwas demonstrated to be significantly inactivated within 24 h, thus restricting the reaction to lowconcentration. Despite this, the resolution method could be used to produce optically pure (+)-1 and endo-(−)-2 from the racemic mixture at 5 g/L substrate. At optimal conditions, 1 g of rac-1 was kinetically resolved to give (+)-1 in 95% ee and 28% yield and endo-(−)-2 in 74% ee, 80% de and 45% yield.
  •  
28.
  • Carlquist, Magnus, et al. (författare)
  • Rationalisation of the substrate concentration dependent diastereoselectivity of a Saccharomyces cerevisiae short-chain dehydrogenase
  • 2007
  • Ingår i: Tetrahedron: Asymmetry. - : Elsevier BV. - 0957-4166. ; 18:21, s. 2554-2556
  • Tidskriftsartikel (refereegranskat)abstract
    • The diastereoselectivity of the carbonyl reduction of bicyclo[2.2.2]octane-2,6-dione, catalysed by the purified yeast cytosolic short-chain dehydrogenase Ymr226cp, was shown to be substrate concentration dependent. The changing selectivity was attributed to two distinct binding configurations of the substrate in the active site, each yielding a distinct hydroxy ketone diastereomer. By applying individual KM and Vmax values for each binding configuration, the concentration dependence could be modelled with Michaelis–Menten kinetics and the apparent KM and Vmax values for the generation of each diastereomer determined. This is to the best of our knowledge the first rationalisation of a substrate dependent stereoselectivity for a pro-chiral substrate with an isolated enzyme.
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29.
  • Friberg, Annika, et al. (författare)
  • Efficient bioreduction of bicyclo[2.2.2]octane-2,5-dione and bicyclo[2.2.2]oct-7-ene-2,5-dione by genetically engineered Saccharomyces cerevisiae
  • 2006
  • Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 4:11, s. 2304-2312
  • Tidskriftsartikel (refereegranskat)abstract
    • A screening of non-conventional yeast species and several Saccharomyces cerevisiae ( baker's yeast) strains overexpressing known carbonyl reductases revealed the S. cerevisiae reductase encoded by YMR226c as highly efficient for the reduction of the diketones 1 and 2 to their corresponding hydroxyketones 3 - 6 ( Scheme 1) in excellent enantiomeric excesses. Bioreduction of 1 using the genetically engineered yeast TMB4100, overexpressing YMR226c, resulted in > 99% ee for hydroxyketone (+)- 4 and 84 - 98% ee for (-)- 3, depending on the degree of conversion. Baker's yeast reduction of diketone 2 resulted in > 98% ee for the hydroxyketones (+)- 5 and (+)- 6. However, TMB4100 led to significantly higher conversion rates ( over 40 fold faster) and also a minor improvement of the enantiomeric excesses (> 99%).
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30.
  • Garcia Sanchez, Rosa, et al. (författare)
  • Cross-reactions between engineered xylose and galactose pathways in recombinant Saccharomyces cerevisiae
  • 2010
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 3:19
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: Overexpression of the PGM2 gene encoding phosphoglucomutase (Pgm2p) has been shown to improve galactose utilization both under aerobic and under anaerobic conditions. Similarly, xylose utilization has been improved by overexpression of genes encoding xylulokinase (XK), enzymes from the non-oxidative pentose phosphate pathway (non-ox PPP) and deletion of the endogenous aldose reductase GRE3 gene in engineered S. cerevisiae strains carrying either fungal or bacterial xylose pathways. In the present study, we investigated how the combination of these traits affect xylose and galactose utilization, in the presence or absence of glucose in S. cerevisiae strains engineered with the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. RESULTS:In the absence of PGM2 overexpression, the combined overexpression of XK, the non-ox PPP and deletion of the GRE3 gene, significantly delayed aerobic growth on galactose, whereas no difference was observed between the control strain and the xylose-engineered strain when the PGM2 gene was overexpressed. Under anaerobic conditions, the overexpression of the PGM2 gene increased the ethanol yield and the xylose consumption rate in medium containing xylose as the only carbon source. The possibility of Pgm2p acting as a xylose isomerase (XI) could be excluded by measuring the XI activity in both strains. The additional copy of the PGM2 gene also resulted in a shorter fermentation time during the co-consumption of galactose and xylose. However, the effect was lost upon addition of glucose to the growth medium. CONCLUSIONS: PGM2 overexpression was shown to benefit xylose and galactose fermentation, alone and in combination. In contrast, galactose fermentation was impaired in the engineered xylose-utilizing strain harbouring extra copies of the non-ox PPP genes and a deletion of the GRE3 gene, unless PGM2 was overexpressed. These cross-reactions are of particular relevance for the fermentation of mixed sugars from lignocellulosic feedstock.
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31.
  • Garcia Sanchez, Rosa, et al. (författare)
  • Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering
  • 2010
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 3
  • Tidskriftsartikel (refereegranskat)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.
  •  
32.
  • Garcia Sanchez, Rosa, et al. (författare)
  • PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae
  • 2010
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 9:1
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: In Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium. RESULTS: In the present study we show that overexpression of PGM2 under control of the HXT7'promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. PGM2 overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When PGM2 was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying PGM2 on the integrative plasmid. CONCLUSION: PGM2 overexpression in S. cerevisiae from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity.
  •  
33.
  • Hahn-Hägerdal, Bärbel, et al. (författare)
  • Bio-ethanol - the fuel of tomorrow from the residues of today
  • 2006
  • Ingår i: Trends in Biotechnology. - : Elsevier BV. - 0167-7799. ; 24:12, s. 549-556
  • Forskningsöversikt (refereegranskat)abstract
    • The increased concern for the security of the oil supply and the negative impact of fossil fuels on the environment, particularly greenhouse gas emissions, has put pressure on society to find renewable fuel alternatives. The most common renewable fuel today is ethanol produced from sugar or grain (starch); however, this raw material base will not be sufficient. Consequently, future large-scale use of ethanol will most certainly have to be based on production from lignocellulosic materials. This review gives an overview of the new technologies required and the advances achieved in recent years to bring lignocellulosic ethanol towards industrial production. One of the major challenges is to optimize the integration of process engineering, fermentation technology, enzyme engineering and metabolic engineering.
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34.
  • Hahn-Hägerdal, Bärbel, et al. (författare)
  • Role of cultivation media in the development of yeast strains for large scale industrial use
  • 2005
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 4:Art. No. 31
  • Forskningsöversikt (refereegranskat)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.
  •  
35.
  • Hahn-Hägerdal, Bärbel, et al. (författare)
  • Towards industrial pentose-fermenting yeast strains
  • 2007
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 74:5, s. 937-953
  • Forskningsöversikt (refereegranskat)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.
  •  
36.
  •  
37.
  • Johanson, Ted, et al. (författare)
  • Identification of a Candida sp reductase behind bicyclic exo-alcohol production
  • 2009
  • Ingår i: Journal of Molecular Catalysis B: Enzymatic. - : Elsevier BV. - 1873-3158 .- 1381-1177. ; 59:4, s. 286-291
  • Tidskriftsartikel (refereegranskat)abstract
    • Stereoselective baker's yeast-catalysed bioreduction of bicyclo [2.2.2]octane-2.6-dione generates (1R, 4S, 6S)-6-hydroxy-bicyclo [2.2.2]octane-2-one (endo-alcohol) with high enantiomeric and diastereomeric excess. In contrast, whole cells and crude membrane fractions of Candida sp. have been reported to produce the unusual (I R, 4S, 6S)-diastereomer (exo-alcohol) as a major product. Previous in silica screening has identified seven membrane or membrane-bound reductases in C albicans as candidates for the exoactivity. In this work, purification of the corresponding exo-reductase(s) as well as the heterologous cloning of the seven candidate genes was attempted in C tropicalis. The overexpression of IPF4033 (AYR1) gene generated an increased exo-to-endo ratio and exo-alcohol production in whole cells and membranes of C tropicalis. In addition, a slight increased exo-to-endo ratio was observed when overexpressing IPF4033 in S. cerevisiae, although the reduction rate and exo-to-endo ratio were several fold lower compared to those obtained with C. tropicalis. (C) 2008 Elsevier B.V. All rights reserved.
  •  
38.
  • Johanson, Ted, et al. (författare)
  • Reaction and strain engineering for improved stereo-selective whole-cell reduction of a bicyclic diketone
  • 2008
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 77:5, s. 1111-1118
  • Tidskriftsartikel (refereegranskat)abstract
    • Reduction of bicyclo[2.2.2]octane-2,6-dione to (1R, 4S, 6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one by whole cells of Saccharomyces cerevisiae was improved using an engineered recombinant strain and process design. The substrate inhibition followed a Han-Levenspiel model showing an effective concentration window between 12 and 22 g/l, in which the activity was kept above 95%. Yeast growth stage, substrate concentration and a stable pH were shown to be important parameters for effective conversion. The over-expression of the reductase gene YDR368w significantly improved diastereoselectivity compared to previously reported results. Using strain TMB4110 expressing YDR368w in batch reduction with pH control, complete conversion of 40 g/l (290 mM) substrate was achieved with 97% diastereomeric excess (de) and >99 enantiomeric excess (ee), allowing isolation of the optically pure ketoalcohol in 84% yield.
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39.
  • Johanson, Ted, et al. (författare)
  • Strain engineering for stereoselective bioreduction of dicarbonyl compounds by yeast reductases
  • 2005
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 5:6-7, s. 513-525
  • Forskningsöversikt (refereegranskat)abstract
    • Pure chiral molecules are needed in the pharmaceutical and chemical industry as intermediates for the production of drugs or fine chemicals. Microorganisms represent an attractive alternative to chemical synthesis since they have the potential to generate single stereoisomers in high enantiomeric excess (ee). The baker's yeast Saccharomyces cerevisiae can notably reduce dicarbonyl compounds (in particular alpha- and beta-diketones and keto esters) to chiral alcohols with high ee. However, products are formed at a low rate. Moreover, large amounts of co-substrate are required for the regeneration of NADPH that is the preferred co-factor in almost all the known dicarbonyl reductions. Traditionally, better ee, reduction rate and product titre have been achieved via process engineering. The advent of recombinant DNA technology provides an alternative strategy to improve productivity and yield by strain engineering. This review discusses two aspects of strain engineering: (i) the generation of strains with higher reductase activity towards dicarbonyl compounds and (ii) the optimisation of co-substrate utilisation for NADPH cofactor regeneration. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
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40.
  • Karhumaa, Kaisa, et al. (författare)
  • Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains
  • 2006
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 5
  • Tidskriftsartikel (refereegranskat)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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41.
  • Karhumaa, Kaisa, et al. (författare)
  • Comparison of xylose fermentation by Saccharomyces cerevisiae strains carrying either xylose reductase and xylitol dehydrogenase or xylose isomerase
  • 2007
  • Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 6:1
  • Tidskriftsartikel (refereegranskat)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.
  •  
42.
  • Karhumaa, Kaisa, et al. (författare)
  • High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae
  • 2007
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 73:5, s. 1039-1046
  • Tidskriftsartikel (refereegranskat)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.
  •  
43.
  • Karhumaa, Kaisa, et al. (författare)
  • Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering
  • 2005
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 22:5, s. 359-368
  • Tidskriftsartikel (refereegranskat)abstract
    • A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae. Copyright (c) 2005 John Wiley & Sons, Ltd.
  •  
44.
  • Karhumaa, Kaisa, et al. (författare)
  • Proteome analysis of the xylose-fermenting mutant yeast strain TMB 3400.
  • 2009
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 26:7, s. 371-382
  • Tidskriftsartikel (refereegranskat)abstract
    • Xylose fermentation in yeast has been a target of research for years, yet not all the factors that may affect xylose fermentation perfomance of yeast strains are known. In this study, the mutant S. cerevisiae strain TMB 3400, which has good xylose fermentation properties, was compared with its parental strain to examine the factors behind the improved xylose utilization at protein level. The proteome of the parental and the mutant strains were characterized by difference in gel electrophoresis (DiGE) to quantitatively identify proteins that are expressed at altered levels in the mutant. The most significant changes detected by proteome analysis were the 6-10-fold increased levels of xylose reductase, xylitol dehydrogenase and transketolase (Tkl1) in the mutant, which is in accordance with previous knowledge about xylose metabolism in yeast. The level of acetaldehyde dehydrogenase (Ald6) was also significantly increased. In addition, several proteins homologous to proteins from yeast species other than S. cerevisiae were identified in both strains, demonstrating the genetic heterogeneity of industrial yeast strains. The results were also compared with a previously reported transcription analysis performed with identical experimental set-up; however, very little correlation between the two datasets was observed. The results of the proteome analysis were in good agreement with a parallel study in which rationally designed overexpression of XR, XDH and the non-oxidative pentose phosphate pathway resulted in similar improvement in xylose utilization, which demonstrates the usefulness of proteome analysis for the identification of target genes for further metabolic engineering strategies in industrial yeast strains. Copyright (c) 2009 John Wiley & Sons, Ltd.
  •  
45.
  • Katz, Michael, et al. (författare)
  • An improved stereoselective reduction of a bicyclic diketone by Saccharomyces cerevisiae combining process optimization and strain engineering
  • 2002
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 59:6, s. 641-648
  • Tidskriftsartikel (refereegranskat)abstract
    • The stereoselective reduction of the bicyclic diketone bicyclo[2.2.2]octane-2,6-dione, to the ketoalcohol (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one, was used as a model reduction to optimize parameters involved in NADPH-dependent reductions in Saccharomyces cerevisiae with glucose as co-substrate. The co-substrate yield (ketoalcohol formed/glucose consumed) was affected by the initial concentration of bicyclic diketone, the ratio of yeast to glucose, the medium composition, and the pH. The reduction of 5 g l(-1) bicyclic diketone was completed in less than 20 h in complex medium (pH 5.5) under oxygen limitation with an initial concentration of 200 g l(-1) glucose and 5 g l(-1) yeast. The co-substrate yield was further enhanced by genetically engineered strains with reduced phosphoglucose isomerase activity and with the gene encoding alcohol dehydrogenase deleted. Co-substrate yields were increased 2.3-fold and 2.4-fold, respectively, in these strains.
  •  
46.
  • Katz, Michael, et al. (författare)
  • Efficient Anaerobic Whole Cell Stereoselective Bioreduction with Recombinant Saccharomyces cerevisiae.
  • 2003
  • Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 84:5, s. 573-582
  • Tidskriftsartikel (refereegranskat)abstract
    • In this study we investigate the NADPH-dependent stereoselective reduction of the bicyclic diketone bicyclo[2.2.2]octane-2,6-dione (BCO2,6D) to the chiral ketoalcohol (1R,4S,6S)-6-hydroxybicyclo[2.2.2]octane-2-one (BCO2one6ol). Our aim was to develop a whole cell batch process for reduction of carbonyl substrates with (i) a high cosubstrate yield (formed product/consumed cosubstrate) and (ii) a high conversion rate under anaerobic conditions with Saccharomyces cerevisiae as biocatalyst and glucose as cosubstrate. Five open reading frames (ORFs), YMR226c, YDR368w, YOR120w, YGL157w, and YGL039w, encoding reductases involved in the conversion of BCO2,6D were identified using cell-free extract from strains belonging to the ExClone collection (yeast ORF expression clones; ResGen, Invitrogen Corp., UK). We report the one-step purification and characterization of three major BCO2,6D reductases, YMR226cp, YDR368wp (YPR1p), and YOR120wp (GCY1p). The reductases were overexpressed under a strong constitutive promoter and the impact on cosubstrate yield, conversion time, glucose consumption rate, and reduction rate was investigated when reductases were overexpressed either alone or in combination with low phosphoglucose isomerase activity (encoded by YBR196c). Combining overexpression of BCO2,6D reductase with reduced glycolytic rate (low phosphoglucose isomerase activity) offers a fast whole cell stereoselective bioreduction system useful for facilitated anaerobic batch conversions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 573-582, 2003.
  •  
47.
  • Katz, Michael, et al. (författare)
  • Mild detergent treatment of Candida tropicalis reveals a NADPH-dependent reductase in the crude membrane fraction, which enables the production of pure bicyclic exo-alcohol
  • 2004
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 21:15, s. 1253-1267
  • Tidskriftsartikel (refereegranskat)abstract
    • This study demonstrated the occurrence of a NADPH-dependent exo-alcohol reductase in the crude membrane fraction of Candida tropicalis. Cytosolic endo-alcohol reductase activity could be separated from the membrane-bound exo-alcohol activity by means of detergent treatment, enabling the preparation of pure exo-alcohol via the enzymatic conversion of the bicyclic diketone, bicyclo[2.2.2]octane-2,6-dione. The exo-alcohol reductase is, to our knowledge, the first membrane-bound NADPH-dependent reductase accepting a xenobiotic carbonyl substrate that was not a steroid. When C. tropicalis was grown on D-sorbitol, a two-fold increase in the exo-reductase activity was observed as compared to when grown on glucose. An in silico comparison at the protein level between putative xenobiotic carbonyl reductases in Candida albicans, C. tropicalis and Saccharomyces cerevisiae was performed to explain why Candida species are often encountered when screening yeasts for novel stereoselective reduction properties. C. albicans contained more reductases with the potential to reduce xenobiotic carbonyl compounds than did S. cerevisiae. C. tropicalis had many membrane-bound reductases (predicted with the bioinformatics program, TMHMM), some of which had no counterpart in the two other organisms. The exo-reductase is suspected to be either a -hydroxysteroid dehydrogenase or a polyol dehydrogenase from either the short chain dehydrogenase family or the dihydroflavonol reductase family.
  •  
48.
  • Katz, Michael, et al. (författare)
  • Screening of two complementary collections of Saccharomyces cerevisiae to identify enzymes involved in stereo-selective reductions of specific carbonyl compounds: an alternative to protein purification.
  • 2003
  • Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 33:2-3, s. 163-172
  • Tidskriftsartikel (refereegranskat)abstract
    • Two collections of Saccharomyces cerevisiae strains, having open reading frames (ORFs) either overexpressed or deleted, were screened to identify cytosolic NADPH-dependent reductases involved in stereo-selective reductions of specific carbonyl compounds. As model compounds diacetyl (diketone) and ethyl acetoacetate (3-oxo ester) were used. The reductases encoded by YBR149w, YDR368w, YMR226c and YOR120w were found to reduce diacetyl and YOL151w, YHR104w, YGL157w, YOR120w and YDR368w reduced ethyl acetoacetate. We cloned YBR149w, YDR368w and YMR226c using a vector with the strong constitutive GPD promotor and investigated the encoded reductases with respect to reduction of diacetyl. YBR149w, YDR368w and YMR226c were shown to encode (S)-specific diacetyl reductases capable of further reducing racemic acetoin to butanediol. Furthermore, we demonstrated that the purified (S)-diacetyl reductase (EC 1.2.1.56) most likely is the Image-arabinose dehydrogenase encoded by YBR149w (Ara1p).
  •  
49.
  • Katzberg, Michael, et al. (författare)
  • Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example
  • 2010
  • Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 11:4, s. 1735-1758
  • Tidskriftsartikel (refereegranskat)abstract
    • The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the gamma-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the gamma-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio-as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.
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50.
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