Development and characterisation of a recombinantSaccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties
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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 p -PsXYL1-ADH1 T andPGK1 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 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.
Key wordsmutagenesis xylose fermentation xylulokinase xylitol dehydrogenase xylose reductase XKS1 XYL1 XYL2 Saccharomyces cerevisiae yeast
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