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

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.

Feasibility of xylose fermentation by engineeredSaccharomyces cerevisiaeoverexpressing endogenous aldose reductase (GRE3), xylitol dehydrogenase (XYL2), and xylulokinase (XYL3) fromScheffersomyces stipitis

“…Our fermentation results by engineered S. cerevisiae strains overexpressing GRE3 are somewhat contradictory to previous reports that S. cerevisiae strains overexpressing GRE3 were not able to ferment xylose efficiently (Richard et al ., ; Toivari et al ., ). As we cannot rule out the possibility that our host strain background (D452‐2) might have caused the opposite outcomes, we cross‐validated our results through introducing the identical cassette expressing GRE3 into S. cerevisiae CEN.PK2‐1D, a laboratory strain that has been used for xylose fermentation study extensively (van Dijken et al ., ; Träff‐Bjerre et al ., ; Toivari et al ., ; Thanvanthri Gururajan et al ., ; Van Vleet et al ., ). After transforming the CEN.PK2‐1D strain by the pSR6‐GX23 plasmid that was used for constructing the DGX23 strain, a transformant was obtained and named CGX23 ( GRE3 , XYL2 , and XYL3 ).…”

Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae

“…[27,31,32] Although the physiological functions of PHO13p are not yet fully understood, the deletion of PHO13 caused significant changes in transcriptional patterns of S. cerevisiae, [33] and reduced accumulation of sedoheptulose, a dead-end metabolite. [21] Other evolutionary engineering studies employed chemical mutagenesis, [34] genome shuffling, [35] genome library screening, [36] or transposon mutagenesis, [37] to create mutant yeast strains possessing improved xylose-utilizing capabilities. Comparative omics analysis including genomic sequencing, transcriptomics, proteomics, metabolomics, or fluxomics were performed to reveal causal genetic elements that enhanced xylose assimilation.…”

“…Other evolutionary engineering studies employed chemical mutagenesis, [ 34 ] genome shuffling, [ 35 ] genome library screening, [ 36 ] or transposon mutagenesis, [ 37 ] to create mutant yeast strains possessing improved xylose‐utilizing capabilities. Comparative omics analysis including genomic sequencing, transcriptomics, proteomics, metabolomics, or fluxomics were performed to reveal causal genetic elements that enhanced xylose assimilation.…”