Putative xylose and arabinose reductases in <i>Saccharomyces cerevisiae</i>

Träff, Karin; Jönsson, Lennart; Hahn‐Hägerdal, Bärbel

doi:10.1002/yea.913

Cited by 79 publications

(46 citation statements)

References 49 publications

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“…The XR activity in the GRE3-overexpressing strain TMB3267 is much lower than in strains overexpressing the P. stipitis XYL1 gene (TMB3120, TMB3001 and TMB3266). We have previously reported XR activity at overexpression of GRE3 with the CUP1 promoter after induction by 0.5 mM Cu 2+ , and the XR activity was then in the same range (0.021 U/mg) as in the present investigation (Träff et al, 2002). In comparison the activity of glucose-6-phosphate dehydrogenase expressed with the same promoter was 1.5 U/mg after growth in the presence of 0.01 mM Cu 2+ (Jeppsson et al 2003a).…”

Section: Role Of Gre3p In Xylose Fermentationsupporting

confidence: 83%

Endogenous NADPH‐dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

Bjerre

Jeppsson

Hahn‐Hägerdal

et al. 2003

Yeast

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Introduction of the xylose pathway from Pichia stipitis into Saccharomyces cerevisiae enables xylose utilization in recombinant S. cerevisiae. However, xylitol is a major by-product. An endogenous aldo-keto reductase, encoded by the GRE3 gene, was expressed at different levels in recombinant S. cerevisiae strains to investigate its effect on xylose utilization. In a recombinant S. cerevisiae strain producing only xylitol dehydrogenase (XDH) from P. stipitis and an extra copy of the endogenous xylulokinase (XK), ethanol formation from xylose was mediated by Gre3p, capable of reducing xylose to xylitol. When the GRE3 gene was overexpressed in this strain, the xylose consumption and ethanol formation increased by 29% and 116%, respectively. When the GRE3 gene was deleted in the recombinant xylose-fermenting S. cerevisiae strain TMB3001 (which possesses xylose reductase and XDH from P. stipitis, and an extra copy of endogenous XK), the xylitol yield decreased by 49% and the ethanol yield increased by 19% in anaerobic continuous culture with a glucose/xylose mixture. Biomass was reduced by 31% in strains where GRE3 was deleted, suggesting that fine-tuning of GRE3 expression is the preferred choice rather than deletion.

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Section: Role Of Gre3p In Xylose Fermentationsupporting

confidence: 83%

Endogenous NADPH‐dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

Bjerre

Jeppsson

Hahn‐Hägerdal

et al. 2003

Yeast

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“…Under some conditions, deletion of GRE3 can decrease xylitol production (42). Two other aldose reductase gene homologs, YPR1 and YJR096w, also encode enzymes requiring NADPH and can utilize xylose as a substrate, but they have much higher K m values for xylose than does Gre3p (29,41). Thus, several genes encoding aldose reductases that could utilize xylose are expressed in S. cerevisiae.…”

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confidence: 99%

Endogenous Xylose Pathway in Saccharomyces cerevisiae

Toivari

Salusjärvi

Ruohonen

et al. 2004

Appl Environ Microbiol

122

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The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter ؊1 h ؊1 and a xylitol yield of 55% when xylose was the main carbon source.The pentose sugar xylose is a major constituent of lignocellulose. Saccharomyces cerevisiae cannot use xylose, instead converting it primarily to xylitol with only a small fraction going into biomass or ethanol (44,45). Recombinant xylose-metabolizing S. cerevisiae strains contain genes from the xylose-utilizing yeast Pichia stipitis coding enzymes for the first two steps in xylose conversion (23,38,46). However, the potential of S. cerevisiae's own enzymes, if they are overexpressed, has not been evaluated.In xylose-utilizing fungi, xylose reductase (XR) reduces xylose to xylitol, which is oxidized to xylulose by xylitol dehydrogenase (XDH). Xylulose is subsequently phosphorylated to xylulose 5-phosphate by xylulokinase and metabolized through the pentose phosphate pathway. S. cerevisiae cannot utilize xylose but can grow on xylulose (15, 43). Thus, the inability of S. cerevisiae to utilize xylose was attributed to its inability to convert xylose to xylulose (15), even though low XR and XDH activities are known in S. cerevisiae (4). A nonspecific aldose reductase, converting xylose to xylitol, was purified and characterized from S. cerevisiae (24); however, the genes coding for the putative XR and XDH enzymes remained unknown. The third enzyme in the xylose pathway, xylulokinase, is encoded by XKS1, a gene that has been cloned from, and probably is functional in, S. cerevisiae (19). Moderate increases in xylulokinase activity are beneficial in recombinant xylose-metabolizing S. cerevisiae strains (9,18,20,21,40).Based on the S. cerevisiae genome sequence (14), the Nterminal amino acid sequence of the previously purified aldoketo reductase corresponds to the open reading frame YHR104w (GRE3), which has 72% amino acid similarity to the XR enzyme of P. stipitis. This enzyme can reduce a wide variety of ketose substrates and requires a NADPH cofactor (24). The XR of P. stipitis can use either NADH or NADPH i...

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“…However, a few have been considered to be of importance during xylose fermentation. The coding sequences and promoters of various redox-related genes, mainly the endogenous aldose reductases (Träff et al, 2002;Träff-Bjerre et al, 2004) were verified but no mutation(s) could be determined.…”

Section: Discussionmentioning

confidence: 99%

Development and characterisation of a recombinantSaccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties

et al. 2007

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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.

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Putative xylose and arabinose reductases in Saccharomyces cerevisiae

Cited by 79 publications

References 49 publications

Endogenous NADPH‐dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

Endogenous NADPH‐dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

Endogenous Xylose Pathway in Saccharomyces cerevisiae

Development and characterisation of a recombinantSaccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties

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