Construction of industrial xylose-fermenting Saccharomyces cerevisiae strains through combined approaches

Xie, Cai-Yun; Yang, Baozhong; Wu, Yan‐Dong; Xia, Zi-Yuan; Gou, Min; Sun, Zhao‐Yong; Tang, Yue‐Qin

doi:10.1016/j.procbio.2020.05.022

Cited by 14 publications

(12 citation statements)

References 42 publications

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“…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/XKS1t-suffix, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were periodically taken to analyze the concentrations of cell, sugar, ethanol, and by-products. XR and XDH activities were assessed according to the method described previously [ 21 ]. All experiments were performed in triplicate.…”

Section: Methodsmentioning

confidence: 99%

“…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%

“…In our previous study, two haploid xylose-fermenting S. cerevisiae strains were constructed by expressing heterologous XR-XDH pathway using a haploid of industrial diploid strain KF7 [ 20 ] as host [ 21 ]. Strain HX57D expressing mutated XR had notably improved xylose consumption rate and ethanol yield compared with HX62W expressing native XR, which demonstrated that expressing mutated XR promoted xylose metabolism in the haploid strain [ 21 ]. In this study, two diploid xylose-fermenting strains were generated from KF7 by overexpressing XR (native or mutated) and XDH from Scheffersomyces stipitis as well as xylulokinase (XK) from S. cerevisiae .…”

Section: Introductionmentioning

confidence: 99%

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

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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Strains and Mediamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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“…Additionally, microbial inhibitory compounds derived from lignin and sugar degradation such as phenolics, furans, and weak acids are released during the chemical pulping process (Pereira et al 2015;He and Chen 2020), which severely impacts the yeasts biocatalyst ability for economical bioconversion of sugars to ethanol (Bhatia et al 2020;Vees et al 2020). Microbial inhibitors have also been shown to negatively impact heterologous xylose metabolism in S. cerevisiae (Deparis et al 2017), with different effects depending on the pathway(s) being expressed (Cunha et al 2019b) and the xylose isomerase (XI) pathway being particularly vulnerable (Xie et al 2020). In addition, the concentrated thick liquor has high viscosity, osmolality, biological oxygen demand and low pH (Pereira et al 2013;Harner et al 2014).…”

Section: Introductionmentioning

confidence: 99%

Adaptation of Saccharomyces cerevisiae in a concentrated spent sulphite liquor waste stream for increased inhibitor resistance

Brandt

García-Aparicio

Görgens

et al. 2021

Appl Microbiol Biotechnol

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The fermentation of spent sulphite liquor (SSL) from the pulping of hardwoods is limited by the combination of xylose, the primary fermentable sugar and high concentrations of microbial inhibitors that decrease the yeast fermentation ability. The inhibitor resistance phenotypes of xylose-capable Saccharomyces cerevisiae strains were therefore enhanced by combining rational engineering for multi-inhibitor tolerance, with adaptation in concentrated hardwood SSL as selective pressure. The adapted strains were assessed in fermentations with 60-80% v/v concentrated SSL under industrially relevant fermentation conditions. During adaptation, strains produced ethanol concentrations between 11.0 and 15.4 g/L in the range of that reported in literature. The adapted TFA40 and TP50 strains displayed enhanced inhibitor resistance phenotypes and were able to ferment xylose-rich SSL at pH below 5, exhibiting improved ethanol yields relative to the reference strain. Using yeast extract and peptone as nitrogen source in concentrated SSL fermentations further improved ethanol yields. However, strains exhibited a trade-off between resistance and ethanol productivity, indicating a carbon/energy cost for the expression of this inhibitor tolerance phenotype. Key points• Achieved fermentation of xylose-rich hardwood spent sulphite liquor at pH below 5.0 • Adaptation of xylose-capable S. cerevisiae in concentrated spent sulphite liquor • Adapted strains exhibited enhanced inhibitor resistance phenotypes.

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Simultaneous identification to monitor consortia strain dynamics of four biofuel yeast species during fermentation

Pérez

Debernardis

Boido

et al. 2020

Journal of Industrial Microbiology and Biotechnology

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Mixed strain dynamics are still not well or easily monitored although recently molecular identification methods have improved our knowledge. This study used a chromogenic differential plating medium that allows the discrimination of four of the main selected biofuel strains that are currently under development for ethanol production from cellulosic hydrolysates. Complete fermentation of hexoses and xylose was obtained with a yeast consortium composed of Spathaspora passalidarum, Scheffersomyces stipitis, Candida akabanensis and Saccharomyces cerevisiae. The results showed that C.akabanensis excessively dominated consortium balance. Reducing its inoculum from 33 to 4.8% improved population strain balance and fermentation efficiency. Comparison of the consortia with single strain fermentations showed that it optimize sugar consumption and ethanol yields. This simple and cheap method also has advantages compared with molecular methods, as the yeast strains do not need to be genetically marked and identified cell proportions are probably active in the fermentation system as compared to DNA determination methods.

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Construction of industrial xylose-fermenting Saccharomyces cerevisiae strains through combined approaches

Cited by 14 publications

References 42 publications

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

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

Adaptation of Saccharomyces cerevisiae in a concentrated spent sulphite liquor waste stream for increased inhibitor resistance

Simultaneous identification to monitor consortia strain dynamics of four biofuel yeast species during fermentation

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