[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to help lay the foundation for further development of xylose-fermentingSaccharomyces 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 endogenousXKS1 xylulokinase gene, were randomly mutagenised to develop improved xylose-fermenting strains. In this study, two gene cassettes (ADH1
) containing the xylose reductase (PsXYL1) and xylitol dehydrogenase (PsXYL2) genes from the xylose-fermenting yeast,Pichia stipitis, were integrated into the genome of a haploidS. cerevisiae strain (CEN.PK 2-1D). The resulting recombinant strain (YUSM 1001) over-expressing theP. stipitis XYL1 andXYL2 genes (but not the endogenousXKS1 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 ofXYL1, XYL2 andXKS1 and theGPD2 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 theXKS1 xylulokinase gene.
Annals of Microbiology 12/2007; 57(4):599-607. DOI:10.1007/BF03175361 · 0.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of a xylose-fermentingSaccharomyces cerevisiae yeast would be of great benefit to the bioethanol industry. The conversion of xylose to ethanol involves a cascade of enzymatic
reactions and processes. Xylose (aldose) reductases catalyse the conversion of xylose to xylitol. The aim of this study was
to clone, characterise and express a cDNA copy of a novel aldose reductase (NCAR-X) from the filamentous fungusNeurospora crassa inS. cerevisiae. NCAR-X harbours an open reading frame (ORF) of 900 nucleotides. This ORF encodes a protein (NCAR-X, assigned NCBI protein accession
ID: XP_956921) consisting of 300 amino acids, with a predicted molecular weight of 34 kDa. TheNCAR-X-encoded aldose reductase showed significant homology to the xylose reductases ofCandida tenuis andPichia stipitis. WhenNCAR-X was expressed under the control of phosphoglycerate kinase I gene (PGK1) regulatory sequences inS. cerevisiae, its expression resulted in the production of biologically active xylose reductase. Small-scale oxygen-limited xylose fermentation
with theNCAR-X containingS. cerevisiae strains resulted in the production of less xylitol and at least 15% more ethanol than the strains transformed with theP. stipitis xylose reductase gene (PsXYL1). TheNCAR-X-encoded enzyme produced byS. cerevisiae was NADPH-dependent and no activity was observed in the presence of NADH. The co-expression of theNCAR-X andPsXYL1 gene constructs inS. cerevisiae constituted an important part of an extensive research program aimed at the development of xylolytic yeast strains capable
of producing ethanol from plant biomass.
Annals of Microbiology 06/2007; 57(2):223-231. DOI:10.1007/BF03175211 · 0.99 Impact Factor
[Show abstract][Hide abstract] 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 recombinantSaccharomyces cerevisiae strain and evaluate the differences in xylose consumption under fermentation conditions.S. cerevisiae YUSM was constitutively catabolite repressed through specific disruptions within theMIG1 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.
Annals of Microbiology 03/2007; 57(1):85-92. DOI:10.1007/BF03175055 · 0.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A pectinolytic industrial yeast strain of Saccharomyces cerevisiae was generated containing the S. cerevisiae endopolygalacturonase gene (PGU1) constitutively expressed under the control of the 3-phosphoglycerate kinase gene (PGK1) promoter. The new strain contains DNA derived exclusively from yeast and expresses a high polygalacturonic acid hydrolyzing activity. Yeast transformation was carried out by an integrative process targeting a dispensable upstream region of the acetolactate synthase locus (ILV2), which determines sulfometuron methyl resistance. Microvinification assays were performed on white and red musts with the transformed UCLMS-1M strain and with the same strain untransformed. It was found that the changes in the pectic polysaccharide contents did not directly affect the taste or flavor of the wine. From the data reported, it is deduced that the chief advantage of using the modified strain is that it improves the yield of must/wine extraction, while it also positively affects some variables relating to appearance.
International Journal of Food Microbiology 08/2005; 102(2):173-83. DOI:10.1016/j.ijfoodmicro.2004.12.012 · 3.08 Impact Factor