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

Vasudevan Thanvanthri Gururajan, Pierre Van Rensburg, Bärbel Hahn-Hägerdal, Isak S. Pretorius, Ricardo R. Cordero Otero

Research output: Contribution to journalArticle

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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 hap-loid 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 (ADHI p-PsXYL1-ADH1T- and PGKlP-PsXYL2-PGK1T) 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) over-expressing 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.

Original languageEnglish
Pages (from-to)599-607
Number of pages9
JournalAnnals of Microbiology
Volume57
Issue number4
Publication statusPublished - 2007

Fingerprint

Xylose
Fermentation
Saccharomyces cerevisiae
Genes
D-Xylulose Reductase
Glycerol
Aldehyde Reductase
Mutagenesis
Ethanol
Yeasts
Catabolite Repression
Glycerolphosphate Dehydrogenase
Metabolic Engineering
Xylitol
Batch Cell Culture Techniques
Pichia
Haploidy
Methane
Growth
Open Reading Frames

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Biotechnology

Cite this

Thanvanthri Gururajan, Vasudevan ; Van Rensburg, Pierre ; Hahn-Hägerdal, Bärbel ; Pretorius, Isak S. ; Cordero Otero, Ricardo R. / Development and characterisation of a recombinant Saccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties. In: Annals of Microbiology. 2007 ; Vol. 57, No. 4. pp. 599-607.
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Development and characterisation of a recombinant Saccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties. / Thanvanthri Gururajan, Vasudevan; Van Rensburg, Pierre; Hahn-Hägerdal, Bärbel; Pretorius, Isak S.; Cordero Otero, Ricardo R.

In: Annals of Microbiology, Vol. 57, No. 4, 2007, p. 599-607.

Research output: Contribution to journalArticle

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AU - Thanvanthri Gururajan, Vasudevan

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AU - Hahn-Hägerdal, Bärbel

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AB - 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 hap-loid 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 (ADHI p-PsXYL1-ADH1T- and PGKlP-PsXYL2-PGK1T) 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) over-expressing 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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