RESUMO
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-ß-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 ß-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 ß-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.
RESUMO
Anaerobic bacteria assimilate cellodextrins from plant biomass by using a phosphorolytic pathway to generate glucose intermediates for growth. The yeast Saccharomyces cerevisiae can also be engineered to ferment cellobiose to ethanol using a cellodextrin transporter and a phosphorolytic pathway. However, strains with an intracellular cellobiose phosphorylase initially fermented cellobiose slowly relative to a strain employing an intracellular ß-glucosidase. Fermentations by the phosphorolytic strains were greatly improved by using cellodextrin transporters with elevated rates of cellobiose transport. Furthermore under stress conditions, these phosphorolytic strains had higher biomass and ethanol yields compared to hydrolytic strains. These observations suggest that, although cellobiose phosphorolysis has energetic advantages, phosphorolytic strains are limited by the thermodynamics of cellobiose phosphorolysis (ΔG°=+3.6kJmol(-1)). A thermodynamic "push" from the reaction immediately upstream (transport) is therefore likely to be necessary to achieve high fermentation rates and energetic benefits of phosphorolysis pathways in engineered S. cerevisiae.
