| 1. |
- Hahn-Hägerdal, Bärbel, et al.
(författare)
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Metabolic engineering for pentose utilization in Saccharomyces cerevisiae
- 2007
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Ingår i: Advances in Biochemical Engineering/Biotechnology. - Berlin, Heidelberg : Springer Berlin Heidelberg. - 0724-6145. - 9783540736509 ; 108, s. 147-177
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The introduction of pentose utilization pathways in baker's yeast Saccharomyces cerevisiae is summarized together with metabolic engineering strategies to improve ethanolic pentose fermentation. Bacterial and fungal xylose and arabinose pathways have been expressed in S. cerevisiae but do not generally convey significant ethanolic fermentation traits to this yeast. A large number of rational metabolic engineering strategies directed among others toward sugar transport, initial pentose conversion, the pentose phosphate pathway, and the cellular redox metabolism have been exploited. The directed metabolic engineering approach has often been combined with random approaches including adaptation, mutagenesis, and hybridization. The knowledge gained about pentose fermentation in S. cerevisiae is primarily limited to genetically and physiologically well-characterized laboratory strains. The translation of this knowledge to strains performing in an industrial context is discussed.
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| 2. |
- Jeppsson, Marie, et al.
(författare)
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The expression of a Pichia stipitis xylose reductase mutant with higher K-M for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae
- 2006
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 93:4, s. 665-673
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Tidskriftsartikel (refereegranskat)abstract
- Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K-M for NADPH (Kostrzynska et al., 1998: FEMS Microbiol Lett 159:107-112) was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source. (c) 2005 Wiley Periodicals, Inc.
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| 3. |
- Almeida, Joao, et al.
(författare)
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Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction.
- 2009
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84, s. 751-761
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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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| 4. |
- Almeida, Joao, et al.
(författare)
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Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae
- 2007
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Ingår i: Journal of Chemical Technology and Biotechnology. - : Wiley. - 0268-2575 .- 1097-4660. ; 82:4, s. 340-349
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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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| 5. |
- Almeida, Joao, et al.
(författare)
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Metabolic effects of furaldehydes and impacts on biotechnological processes.
- 2009
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 82:4, s. 625-638
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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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| 6. |
- Almeida, Joao, et al.
(författare)
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NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae.
- 2008
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 78:6, s. 939-945
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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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| 7. |
- Almeida, Joao, et al.
(författare)
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Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)
- 2008
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1
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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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| 8. |
- Almeida, Joao, et al.
(författare)
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Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate.
- 2009
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Ingår i: Bioresource Technology. - : Elsevier BV. - 1873-2976 .- 0960-8524. ; 100, s. 3674-3677
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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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| 9. |
- Bengtsson, Oskar, et al.
(författare)
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Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae
- 2009
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 2
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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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| 10. |
- Bettiga, Maurizio, et al.
(författare)
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Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway
- 2009
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 8
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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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