Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae

Parachin, Nádia Skorupa; Bergdahl, Basti; Niel, Ed van; Gorwa-Grauslund, Marie-Françoise

doi:10.1016/j.ymben.2011.05.005

Cited by 71 publications

(52 citation statements)

References 55 publications

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“…Transport is a bottleneck in xylose fermentation, at least at low concentrations, and certainly is exacerbated with improvements of intracellular xylose metabolism (15,16,21). Some heterologous xylose transporters have been identified (11,(31)(32)(33), and increased transporter expression has proven to be beneficial for xylose-fermentation performance (17,19,20).…”

Section: Discussionmentioning

confidence: 99%

“…This intrinsic transport is enough to enable growth on xylose and has not limited xylose consumption in early experiments (12). However, with improvements of intracellular xylose conversion being made (e.g., by overexpression of xylulokinase XKS1 and the PPP enzymes) (13,14), transport becomes a bottleneck, especially at low xylose concentrations (15)(16)(17)(18). In several studies, enhanced transport could be used to improve engineered strains further or could be seen as a result of evolutionary engineering (19)(20)(21)(22).…”

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confidence: 99%

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Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Farwick

Bruder

Schadeweg

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

259

347

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All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growthbased screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.xylose transport | major facilitator superfamily | transporter engineering | pentose metabolism | HXT

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Farwick

Bruder

Schadeweg

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

259

347

View full text Add to dashboard Cite

show abstract

“…Thus, many studies attempted to optimize the expression levels of the genes coding for the XR/XDH pathway or to engineer XR and XDH proteins with balanced cofactor preferences to reduce xylitol accumulation and increase the ethanol yield (19,28,(54)(55)(56)(57)(58)(59)(60). While previous efforts to improve xylose fermentation mostly focused on manipulation of enzymatic reactions related to xylose metabolism, this study demonstrated an innovative approach that controls the carbon flux by accumulating the intermediate product (i.e., xylitol) inside the cell to facilitate the reaction toward the target direction.…”

Section: Discussionmentioning

confidence: 99%

Deletion of FPS1 , Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Wei

Kim

et al. 2013

Appl Environ Microbiol

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Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD ؉ / NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

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“…For this reason, prospection of naturally xylose-fermenting yeasts species (e.g., Scheffersomyces stipitis or Pachisolen tannophilus), comparative genomics, and evolutionary analysis have been used as strategies to determine the limiting steps in pentose metabolism [89,90]. Although the identified functional enzymes were expressed in recombinant Saccharomyces cerevisiae industrial strains, their efficiency in fermenting xylose was shown to still be quite low [87,88], suggesting the requirement of additional modifications.…”

Section: Microbial Metabolomicsmentioning

confidence: 99%

“…Although the identified functional enzymes were expressed in recombinant Saccharomyces cerevisiae industrial strains, their efficiency in fermenting xylose was shown to still be quite low [87,88], suggesting the requirement of additional modifications. Overexpression of genes encoding enzymes of non-oxidative pentose phosphate pathway (PPP) [89], replacement of a small amount of enzymes of xylose metabolism [90], as well as isolation of xylose transporters [91,92] were pointed out as crucial factors for the adequate function of this pathway. However, a more holistic approach could also provide the metabolic reconstruction of most biologically relevant and predictive models to improve the fermentative ability of S. cerevisiae [93].…”

Section: Microbial Metabolomicsmentioning

confidence: 99%

Metabolomics applied in bioenergy

Abdelnur

Caldana

Martins

2014

Chem. Biol. Technol. Agric.

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Metabolomics, which represents all the low molecular weight compounds present in a cell or organism in a particular physiological condition, has multiple applications, from phenotyping and diagnostic analysis to metabolic engineering and systems biology. In this review, we discuss the use of metabolomics for selecting microbial strains and engineering novel biochemical routes involved in plant biomass production and conversion. These aspects are essential for increasing the production of biofuels to meet the energy needs of the future. Additionally, we provide a broad overview of the analytic techniques and data analysis commonly used in metabolomics studies.

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Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae

Cited by 71 publications

References 55 publications

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Deletion of FPS1 , Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Metabolomics applied in bioenergy

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