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.

BackgroundThe yeast Saccharomyces cerevisiae is unable to ferment pentose sugars like d-xylose. Through the introduction of the respective metabolic pathway, S. cerevisiae is able to ferment xylose but first utilizes d-glucose before the d-xylose can be transported and metabolized. Low affinity d-xylose uptake occurs through the endogenous hexose (Hxt) transporters. For a more robust sugar fermentation, co-consumption of d-glucose and d-xylose is desired as d-xylose fermentation is in particular prone to inhibition by compounds present in pretreated lignocellulosic feedstocks.ResultsEvolutionary engineering of a d-xylose-fermenting S. cerevisiae strain lacking the major transporter HXT1–7 and GAL2 genes yielded a derivative that shows improved growth on xylose because of the expression of a normally cryptic HXT11 gene. Hxt11 also supported improved growth on d-xylose by the wild-type strain. Further selection for glucose-insensitive growth on d-xylose employing a quadruple hexokinase deletion yielded mutations at N366 of Hxt11 that reversed the transporter specificity for d-glucose into d-xylose while maintaining high d-xylose transport rates. The Hxt11 mutant enabled the efficient co-fermentation of xylose and glucose at industrially relevant sugar concentrations when expressed in a strain lacking the HXT1–7 and GAL2 genes.ConclusionsHxt11 is a cryptic sugar transporter of S. cerevisiae that previously has not been associated with effective d-xylose transport. Mutagenesis of Hxt11 yielded transporters that show a better affinity for d-xylose as compared to d-glucose while maintaining high transport rates. d-glucose and d-xylose co-consumption is due to a redistribution of the sugar transport flux while maintaining the total sugar conversion rate into ethanol. This method provides a single transporter solution for effective fermentation on lignocellulosic feedstocks.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0360-6) contains supplementary material, which is available to authorized users.

Background: Engineering of the yeast Saccharomyces cerevisiae for improved utilization of pentose sugars is vital for cost-efficient cellulosic bioethanol production. Although endogenous hexose transporters (Hxt) can be engineered into specific pentose transporters, they remain subjected to glucose-regulated protein degradation. Therefore, in the absence of glucose or when the glucose is exhausted from the medium, some Hxt proteins with high xylose transport capacity are rapidly degraded and removed from the cytoplasmic membrane. Thus, turnover of such Hxt proteins may lead to poor growth on solely xylose. Results:The low affinity hexose transporters Hxt1, Hxt36 (Hxt3 variant), and Hxt5 are subjected to catabolite degradation as evidenced by a loss of GFP fused hexose transporters from the membrane upon glucose depletion. Catabolite degradation occurs through ubiquitination, which is a major signaling pathway for turnover. Therefore, N-terminal lysine residues of the aforementioned Hxt proteins predicted to be the target of ubiquitination, were replaced for arginine residues. The mutagenesis resulted in improved membrane localization when cells were grown on solely xylose concomitantly with markedly stimulated growth on xylose. The mutagenesis also improved the late stages of sugar fermentation when cells are grown on both glucose and xylose.Conclusions: Substitution of N-terminal lysine residues in the endogenous hexose transporters Hxt1 and Hxt36 that are subjected to catabolite degradation results in improved retention at the cytoplasmic membrane in the absence of glucose and causes improved xylose fermentation upon the depletion of glucose and when cells are grown in d-xylose alone.

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A sandwich-type immunosensor composed of antigen-double target/probe DNA-coated gold nanoparticles (NPs) was developed for the measurement of fluorescence intensity and quantitative analysis of single-stranded DNA based on the concentration of free glyphosate. The reaction between the antigen-double DNA-gold NPs and immobilized antibody on the substrate was carried out for 2 h. The results of the antigen-antibody reaction were measured on the basis of the fluorescence intensity obtained from comparison with the free antigens at concentrations of 0.01-100 μg mL(-1) for the detection of immobilized antigen-double DNA-gold NPs. For the quantitative analysis based on the concentration of glyphosate(0.01-100 μg mL(-1)), the immunosensor response also revealed the same detection range of glyphosate using DNA detection.