Improving pentose fermentation by preventing ubiquitination of hexose transporters in Saccharomyces cerevisiae

Abstract

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.

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

“…Hxt3 is expressed at high glucose concentration but disappears from the membrane at low glucose concentration (Ozcan and Johnston, 1999) via ubiquitination (Snowdon and Merwe, 2012). Recently, we demonstrate that substitution of the N-terminal lysine residues of the endogenous, but fused hexose transporters Hxt36 resulted in improved membrane localization and cell growth on xylose up to the late stage of sugar fermentation (Nijland et al, 2016). Ubiquitylation plays a prominent role in hexose transporters degradation (Horak and Wolf, 1997;Nijland et al, 2016;Roy et al, 2014), whereas phosphorylation is the primary mechanism for regulating cellular signaling (Humphrey et al, 2015).…”

“…Recently, we demonstrate that substitution of the N‐terminal lysine residues of the endogenous, but fused hexose transporters Hxt36 resulted in improved membrane localization and cell growth on xylose up to the late stage of sugar fermentation (Nijland et al, 2016). Ubiquitylation plays a prominent role in hexose transporters degradation (Horak and Wolf, 1997; Nijland et al, 2016; Roy et al, 2014), whereas phosphorylation is the primary mechanism for regulating cellular signaling (Humphrey et al, 2015). However, distinct phosphorylation sites are often used in conjunction with ubiquitylation in degradation, and these distinct sites are more highly conserved than the entire set of phosphorylation sites (Swaney et al, 2013).…”

Substrate-dependent cluster density dynamics in bacterial phosphotransferase system permeases

“…Our data using an N-terminal fluorescent fusion for the fructose specific EIIABC confirms clear membrane localization of the entire complex. It was generally assumed that the EII parts of the PTS are uniformly distributed in the cytoplasmic membrane, similar to other transport proteins, such as the Hxt hexose transporter in yeast (41), and the mentioned BglF (32). However, data on subcellular localization of transport proteins is rather scarce.…”

d‐Xylose consumption by nonrecombinant Saccharomyces cerevisiae: A review

“…The failure of the low-affinity HXT1 and HXT3 permeases to allow growth or fermentation of xylose is probably a consequence of the rapid endocytosis and vacuolar degradation of these transporters in the absence of glucose, even if other carbon sources (galactose, lactate, ethanol, or xylose) are present in the medium (Hovsepian et al, 2017;Nijland et al, 2016;Roy, Kim, Cho, & Kim, 2014). Indeed, in a recombinant hxt-null strain expression of the HXT1 permease allows consumption of xylose during xylose-glucose co-fermentations (but not with xylose alone), and preventing its ubiquitination (and thus endocytosis) also allows growth of the yeast cells on xylose (Gonçalves et al, 2014;Nijland et al, 2016). Nevertheless, recombinant hxt-null strains with genes for xylose metabolism are suitable platforms for cloning and characterization of novel xylose transporters from other xylose-fermenting yeasts (e.g., de Sales et al, 2015;Saloheimo et al, 2007;Young, Poucher, Comer, Bailey, & Alper, 2011).…”