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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

Active site of Cellulomonas uda cellobiose phosphorylase in complex with cellobiose. The crystal structure of C. uda CBP is shown in complex with cellobiose [PDB:3S4A]. CBP is a homodimer whose active sites comprise an (α/α)6-barrel domain of one subunit (blue) and the helical extension from the N-terminal domain of the adjacent subunit (green). Cellobiose is bound in the active site with its reducing end pointing toward the N-terminal extension from the adjacent subunit (green). Arg166 and Gln165 on the adjacent subunit (green) might be in contact with cellobiose bound at the active site of the blue subunit. The 6-methoxy group that is present in cellobiose but absent in the GX molecule is circled. Xylose is expected to bind at the reducing end site, resulting in a possible interaction with the N-terminal extension of the adjacent subunit.
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Figure 7: Active site of Cellulomonas uda cellobiose phosphorylase in complex with cellobiose. The crystal structure of C. uda CBP is shown in complex with cellobiose [PDB:3S4A]. CBP is a homodimer whose active sites comprise an (α/α)6-barrel domain of one subunit (blue) and the helical extension from the N-terminal domain of the adjacent subunit (green). Cellobiose is bound in the active site with its reducing end pointing toward the N-terminal extension from the adjacent subunit (green). Arg166 and Gln165 on the adjacent subunit (green) might be in contact with cellobiose bound at the active site of the blue subunit. The 6-methoxy group that is present in cellobiose but absent in the GX molecule is circled. Xylose is expected to bind at the reducing end site, resulting in a possible interaction with the N-terminal extension of the adjacent subunit.

Mentions: By using in vitro competition assays, we identified xylose as a mixed inhibitor of CBP for the cellobiose phosphorolytic reaction (Figure 3A,B). The synthesis of GX from xylose and G1P, albeit slow[26], shows that xylose can bind to the CBP enzyme active site (Figure 2). This helps to explain the decrease in the apparent affinity for cellobiose in the presence of xylose (increase in KM,app). The decrease in maximal phosphorolytic rate of cellobiose (Vmax, app) in the presence of xylose suggests that xylose also inhibits cellobiose phosphorolytic activity in some other way, unrelated to xylose competition with cellobiose for the enzyme active site. CBP is a homodimer[27,28], and its active site pocket is formed at the interface of an (α/α)6-barrel domain and a helical extension from the N-terminal domain of the adjacent subunit (Figure 7)[28]. Notably, in a crystal structure of Cellulomonas uda CBP in complex with cellobiose [PDB: 3S4A][29], the reducing end of the cellobiose molecule is in contact with the extension from the adjacent subunit (Figure 7). Xylose likely binds at this position, because its structure is similar to that of glucose, enabling the formation of GX from xylose and G1P. Thus, as xylose binds to and/or releases from the reducing end of the active site in one subunit, it may come into contact with the N-terminal domain of the other subunit. The interaction may alter CBP enzymatic activity, preventing cellobiose phosphorylation in the adjacent unit or resulting in decreased product dissociation from the adjacent active site.


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)

Active site of Cellulomonas uda cellobiose phosphorylase in complex with cellobiose. The crystal structure of C. uda CBP is shown in complex with cellobiose [PDB:3S4A]. CBP is a homodimer whose active sites comprise an (α/α)6-barrel domain of one subunit (blue) and the helical extension from the N-terminal domain of the adjacent subunit (green). Cellobiose is bound in the active site with its reducing end pointing toward the N-terminal extension from the adjacent subunit (green). Arg166 and Gln165 on the adjacent subunit (green) might be in contact with cellobiose bound at the active site of the blue subunit. The 6-methoxy group that is present in cellobiose but absent in the GX molecule is circled. Xylose is expected to bind at the reducing end site, resulting in a possible interaction with the N-terminal extension of the adjacent subunit.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4061319&req=5

Figure 7: Active site of Cellulomonas uda cellobiose phosphorylase in complex with cellobiose. The crystal structure of C. uda CBP is shown in complex with cellobiose [PDB:3S4A]. CBP is a homodimer whose active sites comprise an (α/α)6-barrel domain of one subunit (blue) and the helical extension from the N-terminal domain of the adjacent subunit (green). Cellobiose is bound in the active site with its reducing end pointing toward the N-terminal extension from the adjacent subunit (green). Arg166 and Gln165 on the adjacent subunit (green) might be in contact with cellobiose bound at the active site of the blue subunit. The 6-methoxy group that is present in cellobiose but absent in the GX molecule is circled. Xylose is expected to bind at the reducing end site, resulting in a possible interaction with the N-terminal extension of the adjacent subunit.
Mentions: By using in vitro competition assays, we identified xylose as a mixed inhibitor of CBP for the cellobiose phosphorolytic reaction (Figure 3A,B). The synthesis of GX from xylose and G1P, albeit slow[26], shows that xylose can bind to the CBP enzyme active site (Figure 2). This helps to explain the decrease in the apparent affinity for cellobiose in the presence of xylose (increase in KM,app). The decrease in maximal phosphorolytic rate of cellobiose (Vmax, app) in the presence of xylose suggests that xylose also inhibits cellobiose phosphorolytic activity in some other way, unrelated to xylose competition with cellobiose for the enzyme active site. CBP is a homodimer[27,28], and its active site pocket is formed at the interface of an (α/α)6-barrel domain and a helical extension from the N-terminal domain of the adjacent subunit (Figure 7)[28]. Notably, in a crystal structure of Cellulomonas uda CBP in complex with cellobiose [PDB: 3S4A][29], the reducing end of the cellobiose molecule is in contact with the extension from the adjacent subunit (Figure 7). Xylose likely binds at this position, because its structure is similar to that of glucose, enabling the formation of GX from xylose and G1P. Thus, as xylose binds to and/or releases from the reducing end of the active site in one subunit, it may come into contact with the N-terminal domain of the other subunit. The interaction may alter CBP enzymatic activity, preventing cellobiose phosphorylation in the adjacent unit or resulting in decreased product dissociation from the adjacent active site.

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