Abstract
BackgroundEngineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. Consequently, engineered xylose-fermenting yeast strains first utilize D-glucose before D-xylose can be transported and metabolized.ResultsWe have used an evolutionary engineering approach that depends on a quadruple hexokinase deletion xylose-fermenting S. cerevisiae strain to select for growth on D-xylose in the presence of high D-glucose concentrations. This resulted in D-glucose-tolerant growth of the yeast of D-xylose. This could be attributed to mutations at N367 in the endogenous chimeric Hxt36 transporter, causing a defect in D-glucose transport while still allowing specific uptake of D-xylose. The Hxt36-N367A variant transports D-xylose with a high rate and improved affinity, enabling the efficient co-consumption of D-glucose and D-xylose.ConclusionsEngineering of yeast endogenous hexose transporters provides an effective strategy to construct glucose-insensitive xylose transporters that are well integrated in the carbon metabolism regulatory network, and that can be used for efficient lignocellulosic bioethanol production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0168-9) contains supplementary material, which is available to authorized users.
Highlights
Engineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production
Evolutionary engineering of a xylose-metabolizing S. cerevisiae strain in the presence of glucose To select for an improved D-xylose transport in S. cerevisiae, evolutionary engineering was performed with the quadruple hexokinase (GLK1, HXK1, HXK2, and GAL1) deletion strain S. cerevisiae DS71054, which is described in detail elsewhere (Shin et al, submitted)
The strain was used in an evolutionary design experiment to isolate higher affinity D-xylose transporters that are insensitive to D-glucose inhibition
Summary
Engineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. A major issue in the conversion of saccharified cellulosic uptake of D-xylose by the yeast cell [8], while subsequent suboptimal intracellular metabolism and redox balancing hamper the process Some of these issues have been solved by overexpression of the genes of the pentose phosphate pathway [5] (Figure 1) or by the regulation of the redox state during D-xylose fermentation [9]. Mutagenesis of the HXT7 and GAL2 genes yielded a GAL2 mutant that was found to be defective in D-glucose uptake while still retaining substantial D-xylose transport activity [16]
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