Isolation and characterization of a mutant recombinant Saccharomyces cerevisiae strain with high efficiency xylose utilization

Tomitaka, Masataka; Taguchi, Hisataka; Fukuda, Kei; Akamatsu, Takashi; Kida, Kenji

doi:10.1016/j.jbiosc.2013.05.027

“…The pho13 deletion has been reported to improve xylose fermentation and tolerance to toxic chemicals in lignocellulosic hydrolysate [12,13,27,28]. However, the improvement conferred by this deletion was mostly seen in strains harboring an oxidoreductase pathway with only marginally exhibited improvement with xylose isomerase-based strains [13].…”

Section: Resultsmentioning

confidence: 99%

Systematic and evolutionary engineering of a xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields

Lee

¹

,

Jellison

²

,

Alper

³

2014

Biotechnol Biofuels

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BackgroundEfficient xylose fermentation by yeast would improve the economical and sustainable nature of biofuels production from lignocellulosic biomass. However, the efficiency of xylose fermentation by the yeast Saccharomyces cerevisiae is suboptimal, especially in conversion yield, despite decades of research. Here, we present an improved performance of S. cerevisiae in xylose fermentation through systematic and evolutionary engineering approaches.ResultsThe engineering of S. cerevisiae harboring xylose isomerase-based pathway significantly improved the xylose fermentation performance without the need for intensive downstream pathway engineering. This strain contained two integrated copies of a mutant xylose isomerase, gre3 and pho13 deletion and XKS1 and S. stipitis tal1 overexpression. This strain was subjected to rapid adaptive evolution to yield the final, evolved strain (SXA-R2P-E) which could efficiently convert xylose to ethanol with a yield of 0.45 g ethanol/g xylose, the highest yield reported to date. The xylose consumption and ethanol production rates, 0.98 g xylose g cell−1 h−1 and 0.44 g ethanol g cell−1 h−1, respectively, were also among the highest reported. During this process, the positive effect of a pho13 deletion was identified for a xylose isomerase-containing strain and resulted in up to an 8.2-fold increase in aerobic growth rate on xylose. Moreover, these results demonstrated that low inoculum size and the cell transfer at exponential phase was found to be the most effective adaptation strategy during a batch culture adaptation process.ConclusionsThese results suggest that the xylose isomerase pathway should be the pathway of choice for efficient xylose fermentation in S. cerevisiae as it can outperform strains with the oxidoreductase pathway in terms of yield and ethanol production and xylose consumption rates. Consequently, the strain developed in this study could significantly improve the prospect of biofuels production from lignocellulosic biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0122-x) contains supplementary material, which is available to authorized users.

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“…In an effort to further improve xylose consumption rates, we evaluated the impact of a pho13 deletion on the xylose isomerase pathway. The pho13 deletion has been reported to improve xylose fermentation and tolerance to toxic chemicals in lignocellulosic hydrolysate [12,13,27,28]. However, the improvement conferred by this deletion was mostly seen in strains harboring an oxidoreductase pathway with only marginally exhibited improvement with xylose isomerase-based strains [13].…”

Section: The Effect Of Pho13 Deletion With a Xylose Isomerase Pathwaymentioning

confidence: 99%

Systematic and evolutionary engineering of a xylose isomerase-based pathway in

Lee

¹

,

Jellison

²

,

Alper

³

2014

Biotechnol Biofuels

View full text Add to dashboard Cite

BackgroundEfficient xylose fermentation by yeast would improve the economical and sustainable nature of biofuels production from lignocellulosic biomass. However, the efficiency of xylose fermentation by the yeast Saccharomyces cerevisiae is suboptimal, especially in conversion yield, despite decades of research. Here, we present an improved performance of S. cerevisiae in xylose fermentation through systematic and evolutionary engineering approaches.ResultsThe engineering of S. cerevisiae harboring xylose isomerase-based pathway significantly improved the xylose fermentation performance without the need for intensive downstream pathway engineering. This strain contained two integrated copies of a mutant xylose isomerase, gre3 and pho13 deletion and XKS1 and S. stipitis tal1 overexpression. This strain was subjected to rapid adaptive evolution to yield the final, evolved strain (SXA-R2P-E) which could efficiently convert xylose to ethanol with a yield of 0.45 g ethanol/g xylose, the highest yield reported to date. The xylose consumption and ethanol production rates, 0.98 g xylose g cell−1 h−1 and 0.44 g ethanol g cell−1 h−1, respectively, were also among the highest reported. During this process, the positive effect of a pho13 deletion was identified for a xylose isomerase-containing strain and resulted in up to an 8.2-fold increase in aerobic growth rate on xylose. Moreover, these results demonstrated that low inoculum size and the cell transfer at exponential phase was found to be the most effective adaptation strategy during a batch culture adaptation process.ConclusionsThese results suggest that the xylose isomerase pathway should be the pathway of choice for efficient xylose fermentation in S. cerevisiae as it can outperform strains with the oxidoreductase pathway in terms of yield and ethanol production and xylose consumption rates. Consequently, the strain developed in this study could significantly improve the prospect of biofuels production from lignocellulosic biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0122-x) contains supplementary material, which is available to authorized users.

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“…Yeast cells were cultured on YPD20 plates (YP medium (10 g/L yeast extract, 20 g/L peptone) with 20 g/L glucose and 20 g/L agar). Transformants were selected on YPD20 plates supplemented with 100 μg/mL G418 and/or 50 μg/ [52] pKX1(D)X2XK [21] pXDH P TDH3 -XYL2-T TDH3 [52] pXK P TDH3 -XKS1-T TDH3 [52] 19T-xyl1W P TDH3 -XYL1-T TDH3 This study…”

Section: Strains and Mediamentioning

confidence: 99%

“…Three genes XYL1, XYL2, and XKS1 were assembled simultaneously and seamlessly according to the yeast Golden Gate (yGG) method [51] with minor modifications. Native XYL1 gene and mutated XYL1 gene were separately amplified using plasmids pXR [52] and pKX1(D)X2XK [21] as the templates with primer set XYL1t-prefix/XYL1t-suffix. XYL2 gene and XKS1 gene were amplified using plasmids pXDH and pXK [52] as the templates with primer sets XYL2t-prefix/XYL2t-suffix and XKS1t-prefix/XKS1tsuffix, respectively.…”

Section: Construction Of Pk-x1(w)-x2-xk and Pk-x1(d)-x2-xkmentioning

confidence: 99%

“…Native XYL1 gene and mutated XYL1 gene were separately amplified using plasmids pXR [52] and pKX1(D)X2XK [21] as the templates with primer set XYL1t-prefix/XYL1t-suffix. XYL2 gene and XKS1 gene were amplified using plasmids pXDH and pXK [52] as the templates with primer sets XYL2t-prefix/XYL2t-suffix and XKS1t-prefix/XKS1tsuffix, respectively. These primers included overhangs encoding inwardly facing AarI sites separated by four bases from the appropriate yGG-compatible overhangs.…”

Section: Construction Of Pk-x1(w)-x2-xk and Pk-x1(d)-x2-xkmentioning

confidence: 99%

See 1 more Smart Citation

Different transcriptional responses of haploid and diploid S. cerevisiae strains to changes in cofactor preference of XR

Xie

¹

,

Yang

²

,

Song

³

et al. 2020

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Background Xylitol accumulation is a major barrier for efficient ethanol production through heterologous xylose reductase-xylitol dehydrogenase (XR-XDH) pathway in recombinant Saccharomyces cerevisiae. Mutated NADH-preferring XR is usually employed to alleviate xylitol accumulation. However, it remains unclear how mutated XR affects the metabolic network for xylose metabolism. In this study, haploid and diploid strains were employed to investigate the transcriptional responses to changes in cofactor preference of XR through RNA-seq analysis during xylose fermentation. Results For the haploid strains, genes involved in xylose-assimilation (XYL1, XYL2, XKS1), glycolysis, and alcohol fermentation had higher transcript levels in response to mutated XR, which was consistent with the improved xylose consumption rate and ethanol yield. For the diploid strains, genes related to protein biosynthesis were upregulated while genes involved in glyoxylate shunt were downregulated in response to mutated XR, which might contribute to the improved yields of biomass and ethanol. When comparing the diploids with the haploids, genes involved in glycolysis and MAPK signaling pathway were significantly downregulated, while oxidative stress related transcription factors (TFs) were significantly upregulated, irrespective of the cofactor preference of XR. Conclusions Our results not only revealed the differences in transcriptional responses of the diploid and haploid strains to mutated XR, but also provided underlying basis for better understanding the differences in xylose metabolism between the diploid and haploid strains.

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Isolation and characterization of a mutant recombinant Saccharomyces cerevisiae strain with high efficiency xylose utilization

Cited by 11 publications

References 46 publications

Systematic and evolutionary engineering of a xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields

Systematic and evolutionary engineering of a xylose isomerase-based pathway in Saccharomyces cerevisiae for efficient conversion yields

Systematic and evolutionary engineering of a xylose isomerase-based pathway in

Different transcriptional responses of haploid and diploid S. cerevisiae strains to changes in cofactor preference of XR

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