Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae

Abstract

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5'-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K(m) for xylose of 66.7 mM and a specific activity of 1.41 μmol/min/mg. In comparison, the native xylose isomerase was found to have a K(m) for xylose of 117.1 mM and a specific activity of 0.29 μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.

Diversity of phosphorylases in glycoside hydrolase families

“…The first structural determination of the enzyme was reported in Cellvibrio gilvus (Hidaka et al 2006), which was the second structural determination in GH94 after that of N,N'-diacetylchitobiose phosphorylase (Hidaka et al 2004). Cellobiose phosphorylases are often used to increase the efficiency of the fermentation of cellulosic biomass by Saccharomyces caused by intracellular expression of the gene (Aeling et al 2012;Ha et al 2013;Chomvong et al 2014). These enzymes are also used in the production of amylose from cellulosic materials by the concerted action of glycogen phosphorylase (Ohdan et al 2007;You et al 2013).…”

Conversion of various types of lignocellulosic biomass to fermentable sugars using kraft pulping and enzymatic hydrolysis

“…The presence of cellobiose and xylose in hydrolysates of lignocellulosic biomass is not disadvantageous because these sugars may be converted by various microorganisms to value added products, including bioethanol. In contrast to wild-type Saccharomyces cerevisiae strains that cannot produce ethanol from xylose and cellobiose, the engineered strains coferment these sugars (Aeling et al 2012;Hawkins et al 2013). In general, the sugar profiles of the hydrolysates obtained in this study, particularly the high glucose level, enable their application as sugar feedstocks in fermentation processes.…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…In addition, in this study it was reported that heterologous expression of C. stercorarium genes encoding cellobiose phosphorylase (cepA) and LAC12 increased yeast ability to grow on cellobiose (Sadie et al, 2011). Aeling et al (2012) reported that the recombinant S. cerevisiae strain expressing the R. flavefaciens genes encoding xylose isomerase and cellobiose phosphorylase could uptake and assimilate glucose, xylose, and cellobiose under anaerobic conditions (de Haan et al, 2013). Sakamoto et al (2012) developed a recombinant S. cerevisiae strain codisplaying three types of hemicellulolytic enzymes, including endoxylanase from T. reesei, β-xylosidase from A. oryzae, and β-glucosidase from A. aculeatus on the cell-surface.…”