Limits...
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

Model of cellobiose phosphorylase-mediated cellobiose consumption in the presence of xylose. Cellobiose and xylose are simultaneously imported via the cellodextrin transporter CDT-1 (F213L) and endogenous hexose transporters, respectively. Cellobiose undergoes phosphorolytic cleavage via CBP, generating glucose and G1P, both of which enter glycolysis. However, some of the G1P and imported xylose are condensed by CBP to produce GX in its thermodynamically favorable reverse reaction. GX is then transported out of the cell and imported back into the cell by the cellodextrin transporter over the time course of fermentations. The intracellular GX is then cleaved to G1P and xylose by CBP when the intracellular cellobiose concentration drops in later times of the fermentation. Free xylose is then released back into the fermentation broth in the absence of the xylose consumption pathway. CDT-1 (F213L), cellodextrin transporter mutant; Hxt, hexose transporters; CBP, cellobiose phosphorylase; Pi, inorganic phosphate; Glc, glucose; GX, glucopyranosyl-xylose; G1P, glucose-1-phosphate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Model of cellobiose phosphorylase-mediated cellobiose consumption in the presence of xylose. Cellobiose and xylose are simultaneously imported via the cellodextrin transporter CDT-1 (F213L) and endogenous hexose transporters, respectively. Cellobiose undergoes phosphorolytic cleavage via CBP, generating glucose and G1P, both of which enter glycolysis. However, some of the G1P and imported xylose are condensed by CBP to produce GX in its thermodynamically favorable reverse reaction. GX is then transported out of the cell and imported back into the cell by the cellodextrin transporter over the time course of fermentations. The intracellular GX is then cleaved to G1P and xylose by CBP when the intracellular cellobiose concentration drops in later times of the fermentation. Free xylose is then released back into the fermentation broth in the absence of the xylose consumption pathway. CDT-1 (F213L), cellodextrin transporter mutant; Hxt, hexose transporters; CBP, cellobiose phosphorylase; Pi, inorganic phosphate; Glc, glucose; GX, glucopyranosyl-xylose; G1P, glucose-1-phosphate.

Mentions: We propose that, in the presence of xylose, cellobiose is first transported into yeast cells via cellodextrin transporter mutant CDT-1 (F213L) and cleaved by CBP to generate glucose and G1P (Figure 6). At the same time, xylose is transported into the cell via hexose transporters. With xylose and G1P present inside the cell, CBP catalyzes the reverse phosphorolysis reaction, producing GX, which can be exported by the cellodextrin transporter (Figure 6). The model explains the initial decrease in xylose concentration and increase in GX concentration in the media (Figure 1C,E). CDT-1, a proton symporter[22], can reversibly transport substrates[4] because of the thermodynamic driving forces of high intracellular substrate (that is, GX) concentrations competing with high extracellular proton concentrations. At later stages of the fermentation, GX in the media is transported back into the cell via the cellodextrin transporter, along with the rest of the cellobiose (Figure 6). GX is then cleaved by SdCBP to generate G1P and xylose. Some xylose released is then exported back into the media via endogenous hexose transporters. The second part of the model explains the decrease in GX concentration and the increase in xylose concentration in the media (Figure 1C,E).Formation and cleavage of GX as observed here is unfavorable to cellobiose fermentation. GX exhausts resources that could have been dedicated to cellobiose consumption, namely the use of CDT-1 to export and import GX and SdCBP to form and cleave GX. This is especially deleterious because GX processes occur simultaneously with cellobiose consumption (Figures 1A,E and6). Furthermore, GX formation requires G1P as one of the substrates (Figure 2A). G1P is thus diverted from glycolysis, where in the absence of xylose it would be converted to G6P by phosphoglucomutase. GX formation would thereby decrease the rate of ethanol production because some of G1P produced from cellobiose via CBP is wasted in the formation of GX. These considerations suggest that GX formation likely results in a decrease in cellobiose import by CDT-1, slows cleavage of cellobiose, and reduces the rate of ethanol production. Extracellular concentrations of cellobiose and ethanol also support this model (Figure 1A,B).


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)

Model of cellobiose phosphorylase-mediated cellobiose consumption in the presence of xylose. Cellobiose and xylose are simultaneously imported via the cellodextrin transporter CDT-1 (F213L) and endogenous hexose transporters, respectively. Cellobiose undergoes phosphorolytic cleavage via CBP, generating glucose and G1P, both of which enter glycolysis. However, some of the G1P and imported xylose are condensed by CBP to produce GX in its thermodynamically favorable reverse reaction. GX is then transported out of the cell and imported back into the cell by the cellodextrin transporter over the time course of fermentations. The intracellular GX is then cleaved to G1P and xylose by CBP when the intracellular cellobiose concentration drops in later times of the fermentation. Free xylose is then released back into the fermentation broth in the absence of the xylose consumption pathway. CDT-1 (F213L), cellodextrin transporter mutant; Hxt, hexose transporters; CBP, cellobiose phosphorylase; Pi, inorganic phosphate; Glc, glucose; GX, glucopyranosyl-xylose; G1P, glucose-1-phosphate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Model of cellobiose phosphorylase-mediated cellobiose consumption in the presence of xylose. Cellobiose and xylose are simultaneously imported via the cellodextrin transporter CDT-1 (F213L) and endogenous hexose transporters, respectively. Cellobiose undergoes phosphorolytic cleavage via CBP, generating glucose and G1P, both of which enter glycolysis. However, some of the G1P and imported xylose are condensed by CBP to produce GX in its thermodynamically favorable reverse reaction. GX is then transported out of the cell and imported back into the cell by the cellodextrin transporter over the time course of fermentations. The intracellular GX is then cleaved to G1P and xylose by CBP when the intracellular cellobiose concentration drops in later times of the fermentation. Free xylose is then released back into the fermentation broth in the absence of the xylose consumption pathway. CDT-1 (F213L), cellodextrin transporter mutant; Hxt, hexose transporters; CBP, cellobiose phosphorylase; Pi, inorganic phosphate; Glc, glucose; GX, glucopyranosyl-xylose; G1P, glucose-1-phosphate.
Mentions: We propose that, in the presence of xylose, cellobiose is first transported into yeast cells via cellodextrin transporter mutant CDT-1 (F213L) and cleaved by CBP to generate glucose and G1P (Figure 6). At the same time, xylose is transported into the cell via hexose transporters. With xylose and G1P present inside the cell, CBP catalyzes the reverse phosphorolysis reaction, producing GX, which can be exported by the cellodextrin transporter (Figure 6). The model explains the initial decrease in xylose concentration and increase in GX concentration in the media (Figure 1C,E). CDT-1, a proton symporter[22], can reversibly transport substrates[4] because of the thermodynamic driving forces of high intracellular substrate (that is, GX) concentrations competing with high extracellular proton concentrations. At later stages of the fermentation, GX in the media is transported back into the cell via the cellodextrin transporter, along with the rest of the cellobiose (Figure 6). GX is then cleaved by SdCBP to generate G1P and xylose. Some xylose released is then exported back into the media via endogenous hexose transporters. The second part of the model explains the decrease in GX concentration and the increase in xylose concentration in the media (Figure 1C,E).Formation and cleavage of GX as observed here is unfavorable to cellobiose fermentation. GX exhausts resources that could have been dedicated to cellobiose consumption, namely the use of CDT-1 to export and import GX and SdCBP to form and cleave GX. This is especially deleterious because GX processes occur simultaneously with cellobiose consumption (Figures 1A,E and6). Furthermore, GX formation requires G1P as one of the substrates (Figure 2A). G1P is thus diverted from glycolysis, where in the absence of xylose it would be converted to G6P by phosphoglucomutase. GX formation would thereby decrease the rate of ethanol production because some of G1P produced from cellobiose via CBP is wasted in the formation of GX. These considerations suggest that GX formation likely results in a decrease in cellobiose import by CDT-1, slows cleavage of cellobiose, and reduces the rate of ethanol production. Extracellular concentrations of cellobiose and ethanol also support this model (Figure 1A,B).

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