An engineered cryptic Hxt11 sugar transporter facilitates glucose–xylose co-consumption in Saccharomyces cerevisiae

Abstract

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.

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

“…Furthermore, when endocytosis was genetically blocked, GFP tagged Hxt7 and Hxt2 remained plasma membrane localized even at high glucose concentration (Krampe and Boles, 2002; Kruckeberg et al, 1999). The recently characterized Hxt11 transporter appears invariant to high glucose‐induced degradation (Shin et al, 2015), and our data now suggest that this property can be transferred to Hxt2 by substituting its N‐terminus for the N‐tail of Hxt11.…”

“…1) while it is unknown if a similar type of modification occurs. Rather, we previously observed stable Hxt11 membrane expression over a wide range of glucose concentrations (Shin et al, 2015). Possible a lower propensity for modification of this region may render the chimeric Hxt11/2 more stable at the membrane at high sugar concentrations.…”

“…The recently described Hxt11 transporter, however, is stably expressed at the membrane irrespective of the sugar concentration (Shin et al, 2015). Hxt2 is a high capacity glucose transporter that exhibits a high affinity for glucose but that is degraded at high glucose concentrations (Kruckeberg et al, 1999).…”

“…The exact mechanism of high glucose induced degradation of Hxt2 is not known, but for several other Hxt transporters that are subjected to degradation at low glucose concentration, signaling involves ubiquitination of lysine residues at the N‐terminal tail (Roy et al, 2014). Therefore, we hypothesized that when the N‐terminus of Hxt2 is replaced for the corresponding region of Hxt11, stable membrane expression of Hxt2 might be obtained even at high glucose concentration when the gene is expressed under control of the constitutive truncated HXT7 promoter that was also used in previous studies (Shin et al, 2015). The N‐terminal region of Hxt2 and Hxt11 is poorly conserved (Fig.…”

“…Previous studies demonstrated that the conserved asparagine at position 366 of Hxt11 is a key determinant for the sugar specificity (Nijland et al, 2014; Shin et al, 2015). To improve on the xylose transport specificity of the chimeric Hxt11/2 transporter, the HXT2 gene was mutagenized at the corresponding position N361.…”