Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose.

Kulika Chomvong,Jamie H D Cate,Eun Joong Oh,Xin Li,Abigail E Gillespie,Jonathan M Galazka,Stefan Bauer,Yong-Su Jin,Suk-Jin Ha,Vesna Kordić

doi:10.1186/1754-6834-7-85

Abstract

BackgroundCellobiose 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.ResultsYeast 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.ConclusionsXylose 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.

Highlights

Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass
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 Cellobiose phosphorylase (CBP)
Tandem mass spectrometry (MS-MS) further indicated that the product comprised one hexose unit and one pentose unit (Additional file 1: Figure S1B). These results suggested that the in vitro reverse reaction of cellobiose phosphorylase from Saccharophagus degradans (SdCBP) produced a GX dimer when xylose and G1P were provided as substrates

Summary

Introduction

Cellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. The co-fermentation of cellobiose derived from cellulose and xylose derived from hemicellulose allows these sugars to be consumed simultaneously [4], and may enable continuous biofuel production [5]. In this system, cellobiose and xylose are transported into engineered S. cerevisiae using a cellodextrin transporter (that is, CDT1 from Neurospora crassa) and endogenous hexose transporters, respectively [4,6]. A pathway potentially better suited to the anaerobic environment of large-scale biofuels production substitutes cellobiose phosphorolysis for the hydrolytic reaction of βglucosidase [10,11] This pathway comprises cellobiose phosphorylase (CBP), which cleaves intracellular cellobiose into glucose and glucose-1-phosphate (G1P) [12]. The phosphorolytic pathway can be engineered to perform better than the hydrolytic pathway in terms of product yield in stressful conditions like those expected in lignocellulosic hydrolysates [10]

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Discussion

Conclusion