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Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose.

Chomvong K, Kordić V, Li X, Bauer S, Gillespie AE, Ha SJ, Oh EJ, Galazka JM, Jin YS, Cate JH - Biotechnol Biofuels (2014)

Bottom Line: The system generated significant amounts of the byproduct 4-O-β-d-glucopyranosyl-d-xylose (GX), produced by CBP from glucose-1-phosphate and xylose.The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization.Future efforts will require efficient xylose utilization, GX cleavage by a β-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA.

ABSTRACT

Background: Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. Cellobiose phosphorylase (CBP), an intracellular enzyme generally found in anaerobic bacteria, cleaves cellobiose to glucose and glucose-1-phosphate, providing energetic advantages under the anaerobic conditions required for large-scale biofuel production. However, the efficiency of CBP to cleave cellobiose in the presence of xylose is unknown. This study investigated the effect of xylose on anaerobic CBP-mediated cellobiose fermentation by Saccharomyces cerevisiae.

Results: Yeast capable of fermenting cellobiose by the CBP pathway consumed cellobiose and produced ethanol at rates 61% and 42% slower, respectively, in the presence of xylose than in its absence. The system generated significant amounts of the byproduct 4-O-β-d-glucopyranosyl-d-xylose (GX), produced by CBP from glucose-1-phosphate and xylose. In vitro competition assays identified xylose as a mixed-inhibitor for cellobiose phosphorylase activity. The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization. GX was also shown to be a substrate for cleavage by an intracellular β-glucosidase.

Conclusions: Xylose exerted negative impacts on CBP-mediated cellobiose fermentation by acting as a substrate for GX byproduct formation and a mixed-inhibitor for cellobiose phosphorylase activity. Future efforts will require efficient xylose utilization, GX cleavage by a β-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

No MeSH data available.


Related in: MedlinePlus

Fermentation profile of engineered D452-2 and SR8-a strains supplemented with cellobiose and xylose. S. cerevisiae D452-2 (red circle) and SR8-a (orange square) strains were transformed with the pCS plasmid. Anaerobic fermentations were supplied with 80 g/L cellobiose and 40 g/L xylose. Extracellular concentrations of (A) cellobiose, (B) ethanol, (C) xylose, (D) xylitol and (E) glucopyranosyl-xylose are shown. Values and error bars represent the means and standard deviations of two independent biological replicates.
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Figure 4: Fermentation profile of engineered D452-2 and SR8-a strains supplemented with cellobiose and xylose. S. cerevisiae D452-2 (red circle) and SR8-a (orange square) strains were transformed with the pCS plasmid. Anaerobic fermentations were supplied with 80 g/L cellobiose and 40 g/L xylose. Extracellular concentrations of (A) cellobiose, (B) ethanol, (C) xylose, (D) xylitol and (E) glucopyranosyl-xylose are shown. Values and error bars represent the means and standard deviations of two independent biological replicates.

Mentions: Strain SR8-a is an engineered S. cerevisiae strain capable of rapid xylose fermentation[20]. We wondered whether rapid xylose utilization, which would maintain a lower intracellular concentration of xylose, could mitigate formation of GX by CBP. Strain SR8-a (Table 1) and strain D452-2 used above, which lacks a xylose-utilization pathway, were transformed with the pCS plasmid and fermentations were carried out with 80 g/L of cellobiose as a carbon source, with or without 40 g/L of xylose present. In the absence of xylose, the cellobiose consumption and ethanol production profiles of the engineered D452-2 and SR8-a strains were equivalent (Additional file1: Figure S4). Notably, in the presence of xylose, the cellobiose consumption rate of the SR8-a strain was two-fold higher than that of the D452-2 strain (Figure 4A, Table 2). The SR8-a strain completely consumed cellobiose in 40 hours and xylose in 24 hours (Figure 4A,C). In the SR8-a background, GX was produced at a lower concentration, at a slower rate, and started to decrease after 16 hours, in comparison to 48 hours in the D452-2 strain (Figure 4E). In addition, the engineered SR8-a strain produced less xylitol and more ethanol than the D452-2 strain (Figure 4B,D). This result suggested that efficient xylose utilization reduced the formation of GX and increased the CBP-mediated cellobiose consumption rate in the presence of xylose.


Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose.

Chomvong K, Kordić V, Li X, Bauer S, Gillespie AE, Ha SJ, Oh EJ, Galazka JM, Jin YS, Cate JH - Biotechnol Biofuels (2014)

Fermentation profile of engineered D452-2 and SR8-a strains supplemented with cellobiose and xylose. S. cerevisiae D452-2 (red circle) and SR8-a (orange square) strains were transformed with the pCS plasmid. Anaerobic fermentations were supplied with 80 g/L cellobiose and 40 g/L xylose. Extracellular concentrations of (A) cellobiose, (B) ethanol, (C) xylose, (D) xylitol and (E) glucopyranosyl-xylose are shown. Values and error bars represent the means and standard deviations of two independent biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4061319&req=5

Figure 4: Fermentation profile of engineered D452-2 and SR8-a strains supplemented with cellobiose and xylose. S. cerevisiae D452-2 (red circle) and SR8-a (orange square) strains were transformed with the pCS plasmid. Anaerobic fermentations were supplied with 80 g/L cellobiose and 40 g/L xylose. Extracellular concentrations of (A) cellobiose, (B) ethanol, (C) xylose, (D) xylitol and (E) glucopyranosyl-xylose are shown. Values and error bars represent the means and standard deviations of two independent biological replicates.
Mentions: Strain SR8-a is an engineered S. cerevisiae strain capable of rapid xylose fermentation[20]. We wondered whether rapid xylose utilization, which would maintain a lower intracellular concentration of xylose, could mitigate formation of GX by CBP. Strain SR8-a (Table 1) and strain D452-2 used above, which lacks a xylose-utilization pathway, were transformed with the pCS plasmid and fermentations were carried out with 80 g/L of cellobiose as a carbon source, with or without 40 g/L of xylose present. In the absence of xylose, the cellobiose consumption and ethanol production profiles of the engineered D452-2 and SR8-a strains were equivalent (Additional file1: Figure S4). Notably, in the presence of xylose, the cellobiose consumption rate of the SR8-a strain was two-fold higher than that of the D452-2 strain (Figure 4A, Table 2). The SR8-a strain completely consumed cellobiose in 40 hours and xylose in 24 hours (Figure 4A,C). In the SR8-a background, GX was produced at a lower concentration, at a slower rate, and started to decrease after 16 hours, in comparison to 48 hours in the D452-2 strain (Figure 4E). In addition, the engineered SR8-a strain produced less xylitol and more ethanol than the D452-2 strain (Figure 4B,D). This result suggested that efficient xylose utilization reduced the formation of GX and increased the CBP-mediated cellobiose consumption rate in the presence of xylose.

Bottom Line: The system generated significant amounts of the byproduct 4-O-β-d-glucopyranosyl-d-xylose (GX), produced by CBP from glucose-1-phosphate and xylose.The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization.Future efforts will require efficient xylose utilization, GX cleavage by a β-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA.

ABSTRACT

Background: Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. Cellobiose phosphorylase (CBP), an intracellular enzyme generally found in anaerobic bacteria, cleaves cellobiose to glucose and glucose-1-phosphate, providing energetic advantages under the anaerobic conditions required for large-scale biofuel production. However, the efficiency of CBP to cleave cellobiose in the presence of xylose is unknown. This study investigated the effect of xylose on anaerobic CBP-mediated cellobiose fermentation by Saccharomyces cerevisiae.

Results: Yeast capable of fermenting cellobiose by the CBP pathway consumed cellobiose and produced ethanol at rates 61% and 42% slower, respectively, in the presence of xylose than in its absence. The system generated significant amounts of the byproduct 4-O-β-d-glucopyranosyl-d-xylose (GX), produced by CBP from glucose-1-phosphate and xylose. In vitro competition assays identified xylose as a mixed-inhibitor for cellobiose phosphorylase activity. The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization. GX was also shown to be a substrate for cleavage by an intracellular β-glucosidase.

Conclusions: Xylose exerted negative impacts on CBP-mediated cellobiose fermentation by acting as a substrate for GX byproduct formation and a mixed-inhibitor for cellobiose phosphorylase activity. Future efforts will require efficient xylose utilization, GX cleavage by a β-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

No MeSH data available.


Related in: MedlinePlus