Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose.
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 growth-based 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.
Institute of Molecular Biosciences, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
Alexander
A
Farwick
Stefan
S
Bruder
Virginia
V
Schadeweg
Mislav
M
Oreb
Eckhard
E
Boles
D001692:Biological Transport; D005947:Glucose / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
D009004:Monosaccharide Transport Proteins; D014994:Xylose; D005947:Glucose
HXT; major facilitator superfamily; pentose metabolism; transporter engineering; xylose transport