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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.


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Xylose competition assay of Saccharophagus degradans cellobiose phosphorylase activity. The catalytic properties of SdCBP were determined in the presence of 0, 2.5 and 5 mM of xylose. (A) Initial rates of cellobiose phosphorolysis were calculated from the amount of continuous G1P production at different cellobiose concentrations. All reactions were carried out in duplicate. (B) Apparent kinetic parameters of SdCBP supplemented with 0, 2.5 and 5 mM of xylose determined by non-linear curve fitting.
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Figure 3: Xylose competition assay of Saccharophagus degradans cellobiose phosphorylase activity. The catalytic properties of SdCBP were determined in the presence of 0, 2.5 and 5 mM of xylose. (A) Initial rates of cellobiose phosphorolysis were calculated from the amount of continuous G1P production at different cellobiose concentrations. All reactions were carried out in duplicate. (B) Apparent kinetic parameters of SdCBP supplemented with 0, 2.5 and 5 mM of xylose determined by non-linear curve fitting.

Mentions: To investigate the inhibitory effect of xylose on SdCBP activity, the catalytic properties of SdCBP were determined in the presence of varying xylose concentrations (Figure 3), at the time points preceding the production of GX (Additional file1: Figure S3). Initial rates of cellobiose phosphorolysis were calculated from the amount of G1P produced at different cellobiose concentrations (Figure 3A). As the concentration of xylose increased, the apparent maximal rate (Vmax,app) linearly decreased while the apparent substrate concentration at which the reaction rate is half of Vmax,app (KM,app) linearly increased (Figure 3A,B). This result indicated that the apparent affinity of CBP for cellobiose and its maximal phosphorolytic rate for cellobiose were inversely proportional to the xylose concentration, identifying xylose as a mixed-inhibitor for the cellobiose phosphorolysis reaction (Figure 3B). The negative impact of xylose on SdCBP phosphorolytic activity may therefore contribute to the decrease in cellobiose consumption rate observed phenotypically in the above fermentations (Figure 1A), in addition to the production of GX.


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)

Xylose competition assay of Saccharophagus degradans cellobiose phosphorylase activity. The catalytic properties of SdCBP were determined in the presence of 0, 2.5 and 5 mM of xylose. (A) Initial rates of cellobiose phosphorolysis were calculated from the amount of continuous G1P production at different cellobiose concentrations. All reactions were carried out in duplicate. (B) Apparent kinetic parameters of SdCBP supplemented with 0, 2.5 and 5 mM of xylose determined by non-linear curve fitting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Xylose competition assay of Saccharophagus degradans cellobiose phosphorylase activity. The catalytic properties of SdCBP were determined in the presence of 0, 2.5 and 5 mM of xylose. (A) Initial rates of cellobiose phosphorolysis were calculated from the amount of continuous G1P production at different cellobiose concentrations. All reactions were carried out in duplicate. (B) Apparent kinetic parameters of SdCBP supplemented with 0, 2.5 and 5 mM of xylose determined by non-linear curve fitting.
Mentions: To investigate the inhibitory effect of xylose on SdCBP activity, the catalytic properties of SdCBP were determined in the presence of varying xylose concentrations (Figure 3), at the time points preceding the production of GX (Additional file1: Figure S3). Initial rates of cellobiose phosphorolysis were calculated from the amount of G1P produced at different cellobiose concentrations (Figure 3A). As the concentration of xylose increased, the apparent maximal rate (Vmax,app) linearly decreased while the apparent substrate concentration at which the reaction rate is half of Vmax,app (KM,app) linearly increased (Figure 3A,B). This result indicated that the apparent affinity of CBP for cellobiose and its maximal phosphorolytic rate for cellobiose were inversely proportional to the xylose concentration, identifying xylose as a mixed-inhibitor for the cellobiose phosphorolysis reaction (Figure 3B). The negative impact of xylose on SdCBP phosphorolytic activity may therefore contribute to the decrease in cellobiose consumption rate observed phenotypically in the above fermentations (Figure 1A), in addition to the production of GX.

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