Improvement of ethanol productivity during xylose and glucose co-fermentation by xylose-assimilating S. cerevisiae via expression of glucose transporter Sut1

Katahira, Satoshi; Ito, Meguru; Takema, Hisae; Fujita, Yasuya; Tanino, Takanori; Tanaka, Tsutomu; Fukuda, Hideki; Kondo, Akihiko

doi:10.1016/j.enzmictec.2008.03.001

Cited by 113 publications

(69 citation statements)

References 19 publications

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“…Combined, these ORFs comprise the 23 heterologous transporter genes surveyed in this work. Since the SUT1-3 genes of S. stipitis (22,39) are so similar to each other and to C. intermedia GXF1, they were not included in this survey.…”

Section: Methodsmentioning

confidence: 99%

Functional Survey for Heterologous Sugar Transport Proteins, Using Saccharomyces cerevisiae as a Host

Young

Poucher

Comer

et al. 2011

Appl Environ Microbiol

144

136

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Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.

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Section: Methodsmentioning

confidence: 99%

Functional Survey for Heterologous Sugar Transport Proteins, Using Saccharomyces cerevisiae as a Host

Young

Poucher

Comer

et al. 2011

Appl Environ Microbiol

144

136

View full text Add to dashboard Cite

show abstract

“…The predominant transporters in yeast are members of the HXT family (28) and are marked by efficient hexose transport (29) with lower affinities to xylose thus contributing to diauxic growth and flux limitation when attempting pentose utilization in recombinant S. cerevisiae (30). Previous efforts have attempted to identify heterologous transporters with a higher affinity for xylose over glucose (31)(32)(33)(34)(35)(36). However, the vast majority of these transporters are either nonfunctional, not efficient, or not xylose specific (24,37).…”

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confidence: 99%

Rewiring yeast sugar transporter preference through modifying a conserved protein motif

Young¹,

Tong²,

Bui³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

161

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Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe38Ile39Met40, Scheffersomyces stipitis rgt2 Phe38 and Met40, and Saccharomyces cerevisiae hxt7 Ile39Met40Met340 all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties.

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“…However, with improvements of intracellular xylose conversion being made (e.g., by overexpression of xylulokinase XKS1 and the PPP enzymes) (13,14), transport becomes a bottleneck, especially at low xylose concentrations (15)(16)(17)(18). In several studies, enhanced transport could be used to improve engineered strains further or could be seen as a result of evolutionary engineering (19)(20)(21)(22).…”

mentioning

confidence: 99%

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

Farwick

Bruder

Schadeweg

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

268

347

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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

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Improvement of ethanol productivity during xylose and glucose co-fermentation by xylose-assimilating S. cerevisiae via expression of glucose transporter Sut1

Cited by 113 publications

References 19 publications

Functional Survey for Heterologous Sugar Transport Proteins, Using Saccharomyces cerevisiae as a Host

Functional Survey for Heterologous Sugar Transport Proteins, Using Saccharomyces cerevisiae as a Host

Rewiring yeast sugar transporter preference through modifying a conserved protein motif

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

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