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1.
  • Akerberg, C, et al. (författare)
  • Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour
  • 1998
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 49:6, s. 682-690
  • Tidskriftsartikel (refereegranskat)abstract
    • A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 degrees C and pH levels below 6 enhanced the formation of byproducts and D-lactic acid. By-products were formed in the presence of maltose only, whereas D-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 degrees C and 35 degrees C and at pH 6. In the concentration interval studied (up to 180 g l(-1) glucose and 89 g l(-1) lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid.
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2.
  • 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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3.
  • Almeida, Joao, et al. (författare)
  • Developing Saccharomyces cerevisiae strains for second generation bioethanol: Improving xylose fermentation and inhibitor tolerance
  • 2009
  • Ingår i: International Sugar Journal. - 0020-8841. ; 111:1323, s. 172-180
  • Tidskriftsartikel (refereegranskat)abstract
    • The 2nd generation bioethanol will be produced from lignocellulose biomass including agricultural residues such as bagasse, straw, and stover, forest products residues and dedicated energy crops. The bioconversion of lignocellulose raw materials to ethanol requires microorganisms that are able (i) to ferment both hexose and pentose sugars present in lignocellulose and (ii) to tolerate and to remain active in presence of inhibiting compounds generated during lignocellulose pretreatment and hydrolysis. In this review we focus on the development of strains of baker's yeast Saccharomyces cerevisiae. We discuss genetic and metabolic engineering strategies that have been used to improve, independently, xylose fermentation and strain tolerance. Then, we deliberate strategies for simultaneous improvement of xylose utilization and inhibitor tolerance, with the view that a new generation of xylose-utilizing S. cerevisiae strains harboring beneficial traits for xylose consumption and inhibitor tolerance is expected to be available in the foreseeable future.
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4.
  • 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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5.
  • Almeida, João R.M., et al. (författare)
  • Physiological and Molecular Characterization of Yeast Cultures Pre-Adapted for Fermentation of Lignocellulosic Hydrolysate
  • 2023
  • Ingår i: Fermentation. - : MDPI AG. - 2311-5637. ; 9:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Economically feasible bioethanol process from lignocellulose requires efficient fermentation by yeast of all sugars present in the hydrolysate. However, when exposed to lignocellulosic hydrolysate, Saccharomyces cerevisiae is challenged with a variety of inhibitors that reduce yeast viability, growth, and fermentation rate, and in addition damage cellular structures. In order to evaluate the capability of S. cerevisiae to adapt and respond to lignocellulosic hydrolysates, the physiological effect of cultivating yeast in the spruce hydrolysate was comprehensively studied by assessment of yeast performance in simultaneous saccharification and fermentation (SSF), measurement of furaldehyde reduction activity, assessment of conversion of phenolic compounds and genome-wide transcription analysis. The yeast cultivated in spruce hydrolysate developed a rapid adaptive response to lignocellulosic hydrolysate, which significantly improved its fermentation performance in subsequent SSF experiments. The adaptation was shown to involve the induction of NADPH-dependent aldehyde reductases and conversion of phenolic compounds during the fed-batch cultivation. These properties were correlated to the expression of several genes encoding oxidoreductases, notably AAD4, ADH6, OYE2/3, and YML131w. The other most significant transcriptional changes involved genes involved in transport mechanisms, such as YHK8, FLR1, or ATR1. A large set of genes were found to be associated with transcription factors (TFs) involved in stress response (Msn2p, Msn4p, Yap1p) but also cell growth and division (Gcr4p, Ste12p, Sok2p), and these TFs were most likely controlling the response at the post-transcriptional level.
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6.
  • Axelsson, Anders, et al. (författare)
  • Performance of batch and continuous reactors with co-immobilized yeast and beta-galactosidase
  • 1991
  • Ingår i: Journal of Chemical Technology and Biotechnology. - : Wiley. - 0268-2575 .- 1097-4660. ; 52:2, s. 227-241
  • Tidskriftsartikel (refereegranskat)abstract
    • The anaerobic fermentation of deproteinized whey with beta-galactosidase coimmobilized with Saccharomyces cerevisiae in calcium alginate gel beads for the production of ethanol has been studied in a continuous horizontal packed bed reactor (HPBR). The results are compared with batch experiments in a stirred tank reactor. The immobilized yeast cells are exposed to conditions that vary with time and location in the reactor, making a true steady state impossible. In spite of a very low specific growth rate-of the order of 0.01 h-1 in the first section of the HPBR-the yeast cell growth, accompanied by bead expansion in this section, was high enough to create a cell concentration gradient along the reactor. The continuous reactor is preferable to the batch reactor as the galactose conversion is more efficient. The highest volumetric productivity obtained in the HPBR was 125 mol ethanol m-3 h-1 (6 g ethanol dm-3 h-1) at a substrate concentration of 164 mol m-3 lactose (56 g dm-3) and a dilution rate of 0.21 h-1, corresponding to a space velocity of 0.51 dm3 dm-3 gel h-1. The ethanol yield from consumed glucose and galactose was 80%. The ethanol yield from lactose was only 70%, as only 75% of the galactose was consumed while all the lactose and glucose were converted.
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7.
  • Bengtsson, Oskar, et al. (författare)
  • Identification of common traits in improved xylose-growing Saccharomyces cerevisiae for inverse metabolic engineering.
  • 2008
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 25:11, s. 835-847
  • Tidskriftsartikel (refereegranskat)abstract
    • Four recombinant Saccharomyces cerevisiae strains with enhanced xylose growth (TMB3400, C1, C5 and BH42) were compared with two control strains (TMB3399, TMB3001) through genome-wide transcription analysis in order to identify novel targets for inverse metabolic engineering. A subset of 13 genes with changed expression levels in all improved strains was selected for further analysis. Thirteen validation strains and two reference strains were constructed to investigate the effect of overexpressing or deleting these genes in xylose-utilizing S. cerevisiae. Improved aerobic growth rates on xylose were observed in five cases. The strains overexpressing SOL3 and TAL1 grew 19% and 24% faster than their reference strain, and the strains carrying deletions of YLR042C, MNI1 or RPA49 grew 173%, 62% and 90% faster than their reference strain.
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8.
  • 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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9.
  • Bergdahl, Basti, et al. (författare)
  • Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose
  • 2012
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 5:34
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: The concerted effects of changes in gene expression due to changes in the environment are ultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition can therefore give a description of the cellular state with a high degree of functional information. We used this potential to evaluate the metabolic status of two recombinant strains of Saccharomyces cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only in the pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR) and xylitol dehydrogenase (XDH) or the isomerization pathway with xylose isomerase (XI). The isogenic relationship between the two strains ascertains that the observed responses are a result of the particular xylose pathway and not due to unknown changes in regulatory systems. An increased understanding of the physiological state of these strains is important for further development of efficient pentose-utilizing strains for bioethanol production. Results: Using LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites in central carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The general response to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycle intermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strains investigated had significantly different uptake rates of xylose which led to an enhanced response in the XI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stable around 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylate pool. Conclusions: The low uptake of xylose by the XI-strain led to several distinguished responses: depletion of key metabolites in glycolysis and NADPH, a reduced GTP/GDP ratio and accumulation of PEP and aromatic amino acids. These changes are strong indicators of carbon starvation. The XR/XDH-strain displayed few such traits. The coexistence of these traits and a stable adenylate charge indicates that xylose supplies energy to the cells but does not suppress a response similar to carbon starvation. Particular signals may play a role in the latter, of which the GTP/GMP ratio could be a candidate as it decreased significantly in both strains.
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10.
  • 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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11.
  • 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.
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12.
  • 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)
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13.
  • Bon, Elba, et al. (författare)
  • Yeasts in green chemistry
  • 2004
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 5:3, s. 299-300
  • Tidskriftsartikel (refereegranskat)
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14.
  • Carrasco, Cristhian, et al. (författare)
  • SO2-catalyzed steam pretreatment and fermentation of enzymatically hydrolyzed sugarcane bagasse
  • 2010
  • Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 46:2, s. 64-73
  • Tidskriftsartikel (refereegranskat)abstract
    • Sugarcane bagasse is a lignocellulosic residue obtained from sugarcane milling, and a potentially interesting raw material that can be used for fuel ethanol production. In the present study, bagasse was steam pretreated at temperatures between 180 and 205 ◦C, with holding times of 5–10 min using SO2 as a catalyst to determine conditions that provide a good recovery of pentoses and a suitable material for enzymatic hydrolysis. Pretreatment conducted at 190 ◦C for 5 min gave a pentose yield of 57%, with only minor amounts of degradation compounds formed. Commercial cellulolytic enzymes were used to hydrolyze the obtained fiber fractions after pretreatment at different water-insoluble solid contents (2%, 5% and 8% WIS). The overall highest sugar yield achieved from bagasse was 87% at 2% WIS. Fermentation tests were made on both the pentose-rich hemicellulose hydrolysate obtained from the pretreatment, and the enzymatic hydrolysates obtained from the fiber fractions using the xylose-fermenting strain of Saccharomyces cerevisiae TMB3400, as well as the natural xylose-utilizing yeast Pichia stipitis CBS 6054. The pretreatment hydrolysates produced at 2% WIS as well as the enzymatic hydrolysates showed a complete glucose fermentability indicating a low toxicity to the yeasts. The best xylose conversion (more than 60%) was achieved by the strain TMB3400 at 2% WIS.
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15.
  • Eliasson, A., et al. (författare)
  • Cyanide-insensitive respiration.
  • 2003
  • Ingår i: Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology (Springer Lab Manuals). - 3540442154
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
    • Abstract is not available
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16.
  • Fonseca, Alvaro, et al. (författare)
  • Professor Isabel Spencer-Martins 1951-2008 OBITUARY
  • 2008
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 8:7, s. 1208-1208
  • Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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17.
  • Fonseca, Cesar, et al. (författare)
  • L-Arabinose metabolism in Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012: influence of sugar and oxygen on product formation
  • 2007
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 75:2, s. 303-310
  • Tidskriftsartikel (refereegranskat)abstract
    • L-Arabinose utilization by the yeasts Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012 was investigated in aerobic batch cultures and compared, under similar conditions, to D-glucose and D-xylose metabolism. At high aeration levels, only biomass was formed from all the three sugars. When oxygen became limited, ethanol was produced from D-glucose, demonstrating a fermentative pathway in these yeasts. However, pentoses were essentially respired and, under oxygen limitation, the respective polyols accumulated-arabitol from L-arabinose and xylitol from D-xylose. Different L-arabinose concentrations and oxygen conditions were tested to better understand L-arabinose metabolism. P. guilliermondii PYCC 3012 excreted considerably more arabitol from L-arabinose (and also xylitol from D-xylose) than C arabinofermentans PYCC 5603(T). In contrast to the latter, P guilliermondii PYCC 3012 did not produce any traces of ethanol in complex L-arabinose (80 g/l) medium under oxygen-limited conditions. Neither sustained growth nor active metabolism was observed under anaerobiosis. This study demonstrates, for the first time, the oxygen dependence of metabolite and product formation in L-arabinose-assimilating yeasts.
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18.
  • Fonseca, Cesar, et al. (författare)
  • L-Arabinose transport and catabolism in yeast
  • 2007
  • Ingår i: The FEBS Journal. - : Wiley. - 1742-464X. ; 274:14, s. 3589-3600
  • Tidskriftsartikel (refereegranskat)abstract
    • Two yeasts, Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012, which show rapid growth on L-arabinose and very high rates of L-arabinose uptake on screening, were selected for characterization of L-arabinose transport and the first steps of intracellular L-arabinose metabolism. The kinetics of L-arabinose uptake revealed at least two transport systems with distinct substrate affinities, specificities, functional mechanisms and regulatory properties. The L-arabinose catabolic pathway proposed for filamentous fungi also seems to operate in the yeasts studied. The kinetic parameters of the initial L-arabinose-metabolizing enzymes were determined. Reductases were found to be mostly NADPH-dependent, whereas NAD was the preferred cofactor of dehydrogenases. The differences found between the two yeasts agree with the higher efficiency of L-arabinose metabolism in C. arabinofermentans. This is the first full account of the initial steps of L-arabinose catabolism in yeast including the biochemical characterization of a specific L-arabinose transporter.
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19.
  • Fonseca, Cesar, et al. (författare)
  • The glucose/xylose facilitator Gxf1 from Candida intermedia expressed in a xylose-fermenting industrial strain of Saccharomyces cerevisiae increases xylose uptake in SSCF of wheat straw
  • 2011
  • Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 48:6-7, s. 518-525
  • Tidskriftsartikel (refereegranskat)abstract
    • Ethanolic fermentation of lignocellulose raw materials requires industrial xylose-fermenting strains capable of complete and efficient D-xylose consumption. A central question in xylose fermentation by Saccharomyces cerevisiae engineered for xylose fermentation is to improve the xylose uptake. In the current study, the glucose/xylose facilitator Gxf1 from Candida intermedia, was expressed in three different xylose-fermenting S. cerevisiae strains of industrial origin. The in vivo effect on aerobic xylose growth and the initial xylose uptake rate were assessed. The expression of Gxf1 resulted in enhanced aerobic xylose growth only for the TMB3400 based strain. It displayed more than a 2-fold higher affinity for D-xylose than the parental strain and approximately 2-fold higher initial specific growth rate at 4 g/L D-xylose. Enhanced xylose consumption was furthermore observed when the GXF1-strain was assessed in simultaneous saccharification and co-fermentation (SSCF) of pretreated wheat straw. However, the ethanol yield remained unchanged due to increased by-product formation. Metabolic flux analysis suggested that the expression of the Gxf1 transporter had shifted the control of xylose catabolism from transport to the NAD(+) dependent oxidation of xylitol to xylulose. (C) 2011 Elsevier Inc. All rights reserved.
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20.
  • Fonseca, César, et al. (författare)
  • Use of in vivo C-13 nuclear magnetic resonance Spectroscopy to elucidate L-arabinose metabolism in Yeasts
  • 2008
  • Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 74:6, s. 1845-1855
  • Tidskriftsartikel (refereegranskat)abstract
    • Candida arabinofermentans PYCC 5603(T) and Pichia guilliermondii PYCC 3012 were shown to grow well on L-arabinose, albeit exhibiting distinct features that justify an in-depth comparative study of their respective pentose catabolism. Carbon-13 labeling experiments coupled with in vivo NMR were used to investigate L-arabinose metabolism in these yeasts, thereby complementing recently reported physiological and enzymatic data. The label supplied in L-[2-(13)C]arabinose to non-growing cells, under aerobic conditions, was found on C1 and C2 of arabitol and ribitol, C2 of xylitol, and on C1, C2 and C3 of trehalose. The detection of labeled arabitol and xylitol constitutes additional evidence for the operation in yeast of the redox catabolic pathway widespread in filamentous fungi. Furthermore, labeling at positions C1 of trehalose and arabitol demonstrates that glucose-6-phosphate is recycled through the oxidative pentose phosphate pathway (PPP). This result was interpreted as a metabolic strategy to regenerate NADPH, the cofactor essential to sustain L-arabinose catabolism at the level of L-arabinose reductase and L-xylulose reductase. Moreover, the observed synthesis of D-arabitol and ribitol provides a route to supply NAD(+) under oxygen-limiting conditions. In P. guilliermondii PYCC 3012, the strong accumulation of L-arabitol (up to 0.4 M, intracellular concentration) during aerobic L-arabinose metabolism denotes the existence of a bottleneck at the level of L-arabitol 4-dehydrogenase. This report provides the first experimental evidence of a link between L-arabinose metabolism in fungi and the oxidative branch of PPP, and suggests rational guidelines for the design of strategies towards the production of new and efficient L-arabinose-fermenting yeasts.
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21.
  • 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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22.
  • 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.
  •  
23.
  • 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.
  •  
24.
  • 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.
  •  
25.
  • Gárdonyi, Márk, et al. (författare)
  • High capacity xylose transport in Candida intermedia PYCC4715.
  • 2003
  • Ingår i: FEMS Yeast Research. - 1567-1364. ; 3:1, s. 45-52
  • Tidskriftsartikel (refereegranskat)abstract
    • Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (small mu, Greekmax=0.5 h−1, 30°C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km=0.2 mM, Vmax=7.5 mmol h−1 g−1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K=50 mM, Vmax=11 mmol h−1 g−1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.
  •  
26.
  • Gárdonyi, Márk, et al. (författare)
  • The Streptomyces rubiginosus xylose isomerase is missfolded when expressed in Saccharomyces cerevisiae.
  • 2003
  • Ingår i: Enzyme and Microbial Technology. - 0141-0229. ; 32:2, s. 252-259
  • Tidskriftsartikel (refereegranskat)abstract
    • The Streptomyces rubiginosus xylA gene was cloned and expressed in Saccharomyces cerevisiae. No xylose isomerase activity could be detected. The produced xylose isomerase protein was insoluble and could only be recovered from cell lysates by extraction with the detergent sodium dodecyl-sulfate. In contrast, expression of the xylA gene from Thermus thermophilus in the same host strain resulted in soluble xylose isomerase protein with activities of 1 U mg−1 protein. Comparison of available 3D models suggests that the higher number of intra-subunit ion-bridges in the Thermus thermophilus xylose isomerase may stabilise the protein structure and promote folding by Saccharomyces cerevisiae.
  •  
27.
  • Gururajan, Vasudevan Thanvanthri, et al. (författare)
  • Development and characterisation of a recombinant Saccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties
  • 2007
  • Ingår i: Annals of Microbiology. - 1590-4261. ; 57:4, s. 599-607
  • Tidskriftsartikel (refereegranskat)abstract
    • The purpose of this study was to help lay the foundation for further development of xylose-fermenting Saccharomyces cerevisiae yeast strains through an approach that combined metabolic engineering and random mutagenesis in a recombinant haploid strain that overexpressed only two genes of the xylose pathway. Previously, S. cerevisiae strains, overexpressing heterologous genes encoding xylose reductase, xylitol dehydrogenase and the endogenous XKS1 xylulokinase gene, were randomly mutagenised to develop improved xylose-fermenting strains. In this study, two gene cassettes (ADH1(p)-PsXYL1-ADH1(T) and PGK1(p)-PsXYL2-PGK1(T)) containing the xylose reductase (PsXYL1) and xylitol dehydrogenase (PsXYL2) genes from the xylose-fermenting yeast, Pichia stipitis, were integrated into the genome of a haploid S. cerevisiae strain (CEN.PK 2-1D). The resulting recombinant strain (YUSM 1001) overexpressing the P. stipitis XYL1 and XYL2 genes (but not the endogenous XKS1 gene) was subjected to ethyl methane sulfonate (EMS) mutagenesis. The resulting mutants were screened for faster growth rates on an agar medium containing xylose as the sole carbon source. A mutant strain (designated Y-X) that showed 20-fold faster growth in xylose medium in shake-flask cultures was isolated and characterised. In anaerobic batch fermentation, the Y-X mutant strain consumed 2.5-times more xylose than the YUSM 1001 parental strain and also produced more ethanol and glycerol. The xylitol yield from the mutant strain was lower than that from the parental strain, which did not produce glycerol and ethanol from xylose. The mutant also showed a 50% reduction in glucose consumption rate. Transcript levels of XYL1, XYL2 and XKS1 and the GPD2 glycerol 3-phosphate dehydrogenase gene from the two strains were compared with real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. The mutant showed 10-40 times higher relative expression of these four genes, which corresponded with either the higher activities of their encoded enzymes or by-product formation during fermentation. Furthermore, no mutations were observed in the mutant's promoter sequences or the open reading frames of some of its key genes involved in carbon catabolite repression, glycerol production and redox balancing. The data suggest that the enhancement of the xylose fermentation properties of the Y-X mutant was made possible by increased expression of the xylose pathway genes, especially the XKS1 xylulokinase gene.
  •  
28.
  • Görgens, Johann F, et al. (författare)
  • Amino acid supplementation, controlled oxygen limitation and sequential double induction improves heterologous xylanase production by Pichia stipitis
  • 2005
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 5:6-7, s. 677-683
  • Tidskriftsartikel (refereegranskat)abstract
    • Heterologous endo-beta-1,4-xylanase was produced by Pichia stipitis under control of the hypoxia-inducible PsADH2-promoter in a high-cell-clensity culture. After promoter induction by a shift to oxygen limitation, different aeration rates (oxygen transfer rates) were applied while maintaining oxygen-limitation. Initially, enzyme production was higher in oxygen-limited cultures with high rates of oxygen transfer, although the maximum xylanase activity was not significantly influenced. Amino acid supplementation increased the production of the heterologous endo-beta-1,4-xylanase significantly in highly aerated oxygen-limited cultures, until glucose was depleted. A slight second induction of the promoter was observed in all cultures after the glucose had been consumed. The second induction was most obvious in amino acid-supplemented cultures with higher oxygen transfer rates during oxygen limitation. When such oxygen-limited cultures were shifted back to fully aerobic conditions, a significant re-induction of heterologous endo-beta-1,4-xylanase production was observed. Re-induction was accompanied by ethanol consumption. A similar protein production pattern was observed when cultures were first grown on ethanol as sole carbon source and subsequently glucose and oxygen limitation were applied. Thus, we present the first expression system in yeast with a sequential double-inducible promoter. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
  •  
29.
  • Görgens, Johann F, et al. (författare)
  • Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium
  • 2005
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 67:5, s. 684-691
  • Tidskriftsartikel (refereegranskat)abstract
    • Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.
  •  
30.
  • Görgens, Johann F, et al. (författare)
  • Comparison of three expression systems for heterologous xylanase production by S-cerevisiae in defined medium
  • 2004
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 21:14, s. 1205-1217
  • Tidskriftsartikel (refereegranskat)abstract
    • The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 furI autoselection system, significantly improved production levels of heterollogous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated. Copyright (C) 2004 John Wiley Sons, Ltd.
  •  
31.
  • 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.
  •  
32.
  • 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.
  •  
33.
  • Hahn-Hägerdal, Bärbel, et al. (författare)
  • Metabolic engineering of Saccharomyces cerevisiae for xylose utilization.
  • 2001
  • Ingår i: Advances in Biochemical Engineering, Biotechnology. - 0724-6145. ; 73, s. 53-84
  • Tidskriftsartikel (refereegranskat)abstract
    • Metabolic engineering of Saccharomyces cerevisiae for ethanolic fermentation of xylose is summarized with emphasis on progress made during the last decade. Advances in xylose transport, initial xylose metabolism, selection of host strains, transformation and classical breeding techniques applied to industrial polyploid strains as well as modeling of xylose metabolism are discussed. The production and composition of the substrates--lignocellulosic hydrolysates--is briefly summarized. In a future outlook iterative strategies involving the techniques of classical breeding, quantitative physiology, proteomics, DNA micro arrays, and genetic engineering are proposed for the development of efficient xylose-fermenting recombinant strains of S. cerevisiae.
  •  
34.
  • Hahn-Hägerdal, Bärbel, et al. (författare)
  • Microbial pentose metabolism
  • 2004
  • Ingår i: Applied Biochemistry and Biotechnology. - 1559-0291. ; 116:1-3, s. 1207-1209
  • Tidskriftsartikel (refereegranskat)abstract
    • Speakers were asked to provide quantitative data on the performance of different xylose- (or, in one instance, arabinose-) fermenting strains in laboratory media and, where possible, in "industrial" media prepared by hydrolysis of native lignocellulosic substrates. The data provided are presented in Table 1, which gives an indication of the performance of the various strains but should not be taken as a rigorous comparison. In particular, there is considerable variation in the extent of nutritional supplementation of the various media and the degree of detoxification of the hydrolysates. References provided by some of the speakers are also given.
  •  
35.
  • Hahn-Hägerdal, Bärbel, et al. (författare)
  • Production of ethanol from lignocellulosic materials
  • 1988
  • Ingår i: Animal Feed Science and Technology. - : Elsevier BV. - 0377-8401. ; 21:2-4, s. 175-182
  • Tidskriftsartikel (refereegranskat)abstract
    • In the bioconversion of lignocellulosic materials to ethanol, the cost of the raw material and the cost of the enzymes have been identified as the major costs for the product. In addition, many lignocellulosic materials contain considerable amounts of five-carbon sugars which are not easily fermented to ethanol. The possibility of improving the cost-intensive steps was investigated. The steam pre-treatment of a fast-growing species of Salix (Q082) was optimized with respect to treatment time and temperature, to give the best glucose yield after enzymatic hydrolysis. It was found that the semi-continuous production of cellulolytic enzymes with Trichoderem reesei Rutgers C30 in an aqueous two-phase system (an extractive fermentation system) increased the amount of recoverable enzyme activity compared with a batch process. It was also found that the enzyme consumption in the hydrolysis of cellulolytic materials, in filter paper units (FPU) g−1 reducing sugars (RS), could be considerably reduced by recirculating the cellulolytic enzymes in an aqueous two-phase system combined with an ultra-filtration unit. For the fermentation of pentoses, it was found that the combination of the commercial enzyme glucose isomerase and regular bakers' yeast, Saccharomyces cerevisiae, was superior to xylose fermenting yeasts. Yields and productivities comparable to those obtained in hexose fermentation could be achieved.
  •  
36.
  • 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.
  •  
37.
  • 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.
  •  
38.
  • Hahn-Hägerdal, Bärbel, et al. (författare)
  • Welcome to biotechnology for biofuels.
  • 2008
  • Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 1:1
  • Tidskriftsartikel (refereegranskat)
  •  
39.
  •  
40.
  • Hamacher, T, et al. (författare)
  • Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization
  • 2002
  • Ingår i: Microbiology. - 1465-2080. ; 148, s. 2783-2788
  • Tidskriftsartikel (refereegranskat)abstract
    • For an economically feasible production of ethanol from plant biomass by microbial cells, the fermentation of xylose is important. As xylose uptake might be a limiting step for xylose fermentation by recombinant xylose-utilizing Saccharomyces cerevisiae cells a study of xylose uptake was performed. After deletion of all of the 18 hexose-transporter genes, the ability of the cells to take up and to grow on xylose was lost. Reintroduction of individual hexose-transporter genes in this strain revealed that at intermediate xylose concentrations the yeast high- and intermediate-affinity transporters Hxt4, Hxt5, Hxt7 and Ga12 are important xylose-transporting proteins. Several heterologous monosaccharide transporters from bacteria and plant cells did not confer sufficient uptake activity to restore growth on xylose. Overexpression of the xylose-transporting proteins in a xylose-utilizing PUA yeast strain did not result in faster growth on xylose under aerobic conditions nor did it enhance the xylose fermentation rate under anaerobic conditions. The results of this study suggest that xylose uptake does not determine the xylose flux under the conditions and in the yeast strains investigated.
  •  
41.
  •  
42.
  • Hofvendahl, Karin, et al. (författare)
  • Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose
  • 1999
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 51:5, s. 669-672
  • Tidskriftsartikel (refereegranskat)abstract
    • Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase.
  •  
43.
  • Hofvendahl, Karin, et al. (författare)
  • Simultaneous enzymatic wheat starch saccharification and fermentation to lactic acid by Lactococcus lactis
  • 1999
  • Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 52:2, s. 163-169
  • Tidskriftsartikel (refereegranskat)abstract
    • Simultaneous saccharification of starch from whole-wheat flour and fermentation to lactic acid (SSF) was investigated. For saccharification the commercial enzyme mixture SAN Super 240 L, having alpha-amylase, amyloglucosidase and protease activity, was used, and Lactococcus lactis ssp. lactis ATCC 19435 was used for the fermentation. SSF was studied at flour concentrations corresponding to starch concentrations of 90 g/l and 180 g/l and SAN Super concentrations between 3 mu l/g and 8 mu l/g starch. Kinetic models, developed for the saccharification and fermentation, respectively, were used for simulation and data from SSF experiments were used for model verification. The model simulated SSF when sufficient amounts of nutrients were available during fermentation. This was achieved with high wheat hour concentrations or with addition of yeast extract or amino acids. Nutrient release was dependent on the level of enzyme activity.
  •  
44.
  • 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.
  •  
45.
  • 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.
  •  
46.
  • 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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47.
  • 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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48.
  • Johansson, Björn, et al. (författare)
  • Multiple Gene Expression by Chromosomal Integration and CRE-loxP-Mediated Marker Recycling in Saccharomyces cerevisiae
  • 2004
  • Ingår i: Recombinant Gene Expression Reviews and Protocols ( Methods in Molecular Biology ; 267 ). - New Jersey : Humana Press. - 1940-6029 .- 1064-3745. - 9781588292629 - 1592597742 ; 267
  • Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
    • Multiple gene expression can be introduced in a yeast strain with using only two markers by means of the two new vectors described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The gene of interest (YFG1) is cloned between the promoter and terminator of pB3 PGK. The pB3 PGK-YFG1 is integrated into the genome by a single restriction cut within the YFG1 gene and integrated in the YFG1 locus. The strain is further transformed with the pCRE3 vector. The CRE recombinase expressed from this vector removes the zeocin marker and makes it possible to use the pB3 PGK vector over again in the same strain after curing of the pCRE3 vector. The 2µ-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 is easily cured by growth in nonselective medium without active counterselection. The screening for loss of the chromosomal zeocin marker, as well as curing of the pCRE3 vector, is done in one step, by scoring zeocin sensitivity. This can be done because the zeocin marker is present in both the pB3 PGK and pCRE3. The S. cerevisiae pentose phosphate pathway genes RK11, RPE1, TAL1, and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in simultaneous overexpression of the genes in the xylose-fermenting S. cerevisiae strain TMB3001.
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49.
  • Johansson, Björn, et al. (författare)
  • Overproduction of pentose phosphate pathway enzymes using a new CRE-loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae
  • 2002
  • Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 19:3, s. 225-231
  • Tidskriftsartikel (refereegranskat)abstract
    • Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3. The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counter-selection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.
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50.
  • Johansson, Björn, et al. (författare)
  • The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001
  • 2002
  • Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1364 .- 1567-1356. ; 2:3, s. 277-282
  • Tidskriftsartikel (refereegranskat)abstract
    • Saccharomyces cerevisiae is able to ferment xylose, when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However, xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed to have an insufficient capacity of the non-oxidative pentose phosphate pathway (PPP) for rapid xylose fermentation. Strains overproducing the non-oxidative PPP enzymes ribulose 5-phosphate epimerase (EC 5.1.3.1), ribose 5-phosphate ketol isomerase (EC 5.3.1.6), transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1), as well as all four enzymes simultaneously, were compared with respect to xylose and xylulose fermentation with their xylose-fermenting predecessor S. cerevisiae TMB3001, expressing XYL1, XYL2 and only overexpressing XKS1 (xylulokinase). The level of overproduction in S. cerevisiae TMB3026, overproducing all four non-oxidative PPP enzymes, ranged between 4 and 23 times the level in TMB001. Overproduction of the non-oxidative PPP enzymes did not influence the xylose fermentation rate in either batch cultures of 50 g l(-1) xylose or chemostat cultures of 20 g l(-1) glucose and 20 g l(-1) xylose. The low specific growth rate on xylose was also unaffected. The results suggest that neither of the non-oxidative PPP enzymes has any significant control of the xylose fermentation rate in S. cerevisiae TM133001. However, the specific growth rate on xylulose increased from 0.02-0.03 for TMB3001 to 0.12 for the strain overproducing only transaldolase (TAL1) and to 0.23 for TMB3026, suggesting that overproducing all four enzymes has a synergistic effect. TMB3026 consumed xylulose about two times faster than TMB30001 in batch culture of 50 g l(-1) xylulose. The results indicate that growth on xylulose and the xylulose fermentation rate are partly controlled by the non-oxidative PPP, whereas control of the xylose fermentation rate is situated upstream of xylulokinase, in xylose transport, in xylose reductase, and/or in the xylitol dehydrogenase. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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