Engineering of yeast hexose transporters to transport d -xylose without inhibition by d -glucose

Abstract

All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growthbased screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.xylose transport | major facilitator superfamily | transporter engineering | pentose metabolism | HXT

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

“…A possible solution for the xylose transport dilemma is the engineering of endogenous Hxt transporters to make them more specific for xylose transport (Farwick et al, 2014; Young et al, 2014). This can be achieved through mutagenesis of a highly conserved asparagine residue that is part of the sugar binding site.…”

Enhanced Production of Bioethanol by Fermentation of Autohydrolyzed and C4mimOAc-Treated Sugarcane Bagasse Employing Various Yeast Strains

“…ATCC strain S. stipitis is considered as comparatively more efficient in fermenting xylose to ethanol, but its lower production with all pretreated samples can be attributed to its less efficient glucose utilization as compared to S. cerevisiae [24]. Moreover, it was also suggested in previous studies that all symporters (i.e., the proteins that assist in passage of molecules through the plasma membrane) in S. stipitis are competitively inhibited by glucose molecules, which makes it difficult to utilize both sugars simultaneously, hindering the production of ethanol from xylose [25]. The difference in amount of ethanol produced by these strains can be partially attributed to their difference in tolerance against side-products released during different pretreatment conditions and enzymatic hydrolysis.…”

Enhancement of Simultaneous Xylose and Glucose Utilization by Regulating ZWF1 and PGI1 in Saccharomyces Cerevisiae

“…Therefore, several strategies have been applied to achieve simultaneous xylose and glucose utilization [4]. Xylose-specific transporters and high-affinity transporters of xylose were developed by evolution of xylose-utilizing yeast, directed evolution or rational design of glucose transporters [23][24][25][26][27]. Except for transporter engineering, reconstitution of carbohydrate metabolic networks, such as the phosphoketolase pathway, is another approach to improve xylose utilization [15,28].…”