Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications

Abstract

Lignocellulosic biomass yields after hydrolysis, besides the hexose D-glucose, D-xylose, and L-arabinose as main pentose sugars. In second generation bioethanol production utilizing the yeast Saccharomyces cerevisiae, it is critical that all three sugars are co-consumed to obtain an economically feasible and robust process. Since S. cerevisiae is unable to metabolize pentose sugars, metabolic pathway engineering has been employed to introduce the respective pathways for D-xylose and L-arabinose metabolism. However, S. cerevisiae lacks specific pentose transporters, and these sugars enter the cell with low affinity via glucose transporters of the Hxt family. Therefore, in the presence of D-glucose, utilization of D-xylose and L-arabinose is poor as the Hxt transporters prefer D-glucose. To solve this problem, heterologous expression of pentose transporters has been attempted but often with limited success due to poor expression and stability, and/or low turnover. A more successful approach is the engineering of the endogenous Hxt transporter family and evolutionary selection for D-glucose insensitive growth on pentose sugars. This has led to the identification of a critical and conserved asparagine residue in Hxt transporters that, when mutated, reduces the D-glucose affinity while leaving the D-xylose affinity mostly unaltered. Likewise, mutant Gal2 transporter have been selected supporting specific uptake of L-arabinose. In fermentation experiments, the transporter mutants support efficient uptake and consumption of pentose sugars, and even co-consumption of D-xylose and D-glucose when used at industrial concentrations. Further improvements are obtained by interfering with the post-translational inactivation of Hxt transporters at high or low D-glucose concentrations. Transporter engineering solved major limitations in pentose transport in yeast, now allowing for co-consumption of sugars that is limited only by the rates of primary metabolism. This paves the way for a more economical second-generation biofuels production process.

Biological and Pharmacological Potential of Xylitol: A Molecular Insight of Unique Metabolism

“…Xylose is taken up by cells with the help of specific or non-specific sugar transporters depending on the microbe, e.g., Candida species uses two types of transporter system acting separately depending on the physiological environment: under favorable conditions, cells uptake xylose via carrier-mediated facilitated diffusion transporter, while proton symporter operates under nutrition depleted conditions. Contrary to Candida spp., no such separate xylose-specific transporters have been reported from Saccharomyces cerevisiae, which imports xylose by both high-as well as low-affinity glucose transporters [43]. The final yield of xylitol was lowered due to the simultaneous utilization of xylose in central cellular metabolism for microbial growth and energy generation along with xylitol production.…”

Optimization of 1,2,4‐butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance

“…This result suggests the BT titer in S. cerevisiae can be further increased with long-time cultivation. Furthermore, to obtain high yields of BT, glucose and xylose should be simultaneously consumed, for example, by engineering the hexose transporter or expressing heterologous xylose transporter (Farwick, Bruder, Schadeweg, Oreb, & Boles, 2014;Nijland & Driessen, 2020).…”

Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast

“…xylose facilitators, with no glucose uptake activity, and Xut1 is able to transport glucose, but with lower affinity. However, the native hexose transporters showed the highest xylose uptake activities, even when compared with xylose-specific transporters, like CiGsx1 (F38I39M40), SsXyp29, NcAn25, and SsXut1 [11,12]. Metabolic engineering strategies to reduce endogenous hexose transporter affinity for glucose or to raise the affinity for xylose could be the possible ways to improve ethanol production during mixed sugar fermentations.…”