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Sökning: WFRF:(Otero Ricardo Cordero)

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1.
  • 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.
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2.
  • Lönn, Anna, et al. (författare)
  • Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.
  • 2002
  • Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956. ; 269:1, s. 157-163
  • Tidskriftsartikel (refereegranskat)abstract
    • Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.
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3.
  • Thanvanthri, Vasu, et al. (författare)
  • A constitutive catabolite repression mutant of a recombinant Saccharomyces cerevisiae strain improves xylose consumption during fermentation
  • 2007
  • Ingår i: Annals of Microbiology. - 1590-4261. ; 57:1, s. 85-92
  • Tidskriftsartikel (refereegranskat)abstract
    • Efficient xylose utilisation by microorganisms is of importance to the lignocellulose fermentation industry. The aim of this work was to develop constitutive catabolite repression mutants in a xylose-utilising recombinant Saccharomyces cerevisiae strain and evaluate the differences in xylose consumption under fermentation conditions. S. cerevisiae YUSM was constitutively catabolite repressed through specific disruptions within the MIG1 gene. The strains were grown aerobically in synthetic complete medium with xylose as the sole carbon source. Constitutive catabolite repressed strain YCR17 grew four-fold better on xylose in aerobic conditions than the control strain YUSM. Anaerobic batch fermentation in minimal medium with glucose-xylose mixtures and N-limited chemostats with varying sugar concentrations were performed. Sugar utilisation and metabolite production during fermentation were monitored. YCR17 exhibited a faster xylose consumption rate than YUSM under high glucose conditions in nitrogen-limited chemostat cultivations. This study shows that a constitutive catabolite repressed mutant could be used to enhance the xylose consumption rate even in the presence of high glucose in the fermentation medium. This could help in reducing fermentation time and cost in mixed sugar fermentation.
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