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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 strain D452-2 in the presence and absence of xylose.S. cerevisiae strain D452-2 was transformed with the pCS plasmid, encoding cellodextrin transporter cdt1-F213L and SdCBP. Anaerobic fermentations were supplied with 80 g/L cellobiose in the presence (red dots) and absence (blue dots) of 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 1: Fermentation profile of engineered strain D452-2 in the presence and absence of xylose.S. cerevisiae strain D452-2 was transformed with the pCS plasmid, encoding cellodextrin transporter cdt1-F213L and SdCBP. Anaerobic fermentations were supplied with 80 g/L cellobiose in the presence (red dots) and absence (blue dots) of 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: A codon-optimized CBP gene from Saccharophagus degradans (SdCBP)[10] and a mutant cellodextrin transporter encoding N. crassa CDT-1 (F213L)[10] were cloned into the 2μ plasmid pRS426 under the control of constitutive PPGK1 promoters (hereafter called pCS plasmid). S. cerevisiae strain D452-2 transformed with this plasmid was used for anaerobic fermentations (Table 1). The fermentations were carried out with 80 g/L of cellobiose as a carbon source, either with or without 40 g/L of xylose present. The engineered strain was capable of fermenting cellobiose to ethanol in both conditions (Figure 1A,B). However, in the presence of xylose, the rates of cellobiose consumption and ethanol production decreased by 61% and 42%, respectively (Figure 1A,B, Table 2). As a result, approximately 20 g/L of cellobiose remained in the fermentation broth after 72 hours in the presence of xylose (Figure 1A), whereas all of the cellobiose was consumed within 36 hours in the absence of xylose (Figure 1A). These results indicated that the presence of xylose had a severely negative impact on cellobiose fermentation mediated by CBP.Interestingly, in the fermentation supplied with 40 g/L of xylose, the xylose concentration showed an initial decrease followed by a slight recovery after 36 hours (Figure 1C). Xylitol was also produced with a titer of approximately 9 g/L at 72 hours (Figure 1D). These results suggest that approximately half of the xylose transported into the cell was reduced to xylitol but the rest remained unaccounted for.


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 strain D452-2 in the presence and absence of xylose.S. cerevisiae strain D452-2 was transformed with the pCS plasmid, encoding cellodextrin transporter cdt1-F213L and SdCBP. Anaerobic fermentations were supplied with 80 g/L cellobiose in the presence (red dots) and absence (blue dots) of 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 1: Fermentation profile of engineered strain D452-2 in the presence and absence of xylose.S. cerevisiae strain D452-2 was transformed with the pCS plasmid, encoding cellodextrin transporter cdt1-F213L and SdCBP. Anaerobic fermentations were supplied with 80 g/L cellobiose in the presence (red dots) and absence (blue dots) of 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: A codon-optimized CBP gene from Saccharophagus degradans (SdCBP)[10] and a mutant cellodextrin transporter encoding N. crassa CDT-1 (F213L)[10] were cloned into the 2μ plasmid pRS426 under the control of constitutive PPGK1 promoters (hereafter called pCS plasmid). S. cerevisiae strain D452-2 transformed with this plasmid was used for anaerobic fermentations (Table 1). The fermentations were carried out with 80 g/L of cellobiose as a carbon source, either with or without 40 g/L of xylose present. The engineered strain was capable of fermenting cellobiose to ethanol in both conditions (Figure 1A,B). However, in the presence of xylose, the rates of cellobiose consumption and ethanol production decreased by 61% and 42%, respectively (Figure 1A,B, Table 2). As a result, approximately 20 g/L of cellobiose remained in the fermentation broth after 72 hours in the presence of xylose (Figure 1A), whereas all of the cellobiose was consumed within 36 hours in the absence of xylose (Figure 1A). These results indicated that the presence of xylose had a severely negative impact on cellobiose fermentation mediated by CBP.Interestingly, in the fermentation supplied with 40 g/L of xylose, the xylose concentration showed an initial decrease followed by a slight recovery after 36 hours (Figure 1C). Xylitol was also produced with a titer of approximately 9 g/L at 72 hours (Figure 1D). These results suggest that approximately half of the xylose transported into the cell was reduced to xylitol but the rest remained unaccounted for.

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