Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

Abstract

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (V_max) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

Keywords: evolutionary engineering; sugar transporter; transcriptome; xylose transport; yeast.

FIG 1

Growth of the original DS69473 strain (closed symbols) and the DS69473Evo strain (open symbols) on 2% d-xylose and 0% (■ and □), 6% (● and ○), and 12% (▲ and △) d-glucose.

FIG 2

d-Xylose uptake by the in vivo engineered S. cerevisiae strain. Uptake of 100 mM d-[¹⁴C]xylose by the DS69473 (□) and DS69473Evo (■) strains in the presence of competing concentrations of d-glucose ranging from 0 to 800 mM. (inset) Xylose uptake normalized to the rate observed in the absence of competing glucose.

FIG 3

Growth of the DS69473-Y353 strain (■) and the DS69473-Y353C strain (□) on 2% d-xylose and 12% d-glucose.

FIG 4

Kinetic parameters for d-xylose (A) and d-glucose (B) uptake. Uptake was measured in nmol/mg (dry weight) · min in the DS69473-Y353 (□) and DS69473-Y353C (■) strains. The uptake levels of both sugars for the DS68625 strain, in which HXT1 to HXT7 and GAL2 were deleted, were subtracted from those of the DS69473 and DS69473-Y353C strains to correct for background sugar uptake and cellular binding.