Sugar transport sequences, yeast strains having improved sugar uptake, and methods of use
Inventors
Jeffries, Thomas William • Bae, JuYun • Lin, Bernice Chin-yun • Van Vleet, Jennifer Rebecca Headman
Assignees
US Department of Agriculture USDA • Wisconsin Alumni Research Foundation
Publication Number
US-9150869-B2
Publication Date
2015-10-06
Expiration Date
2029-06-15
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Abstract
Disclosed are nucleic acid constructs comprising coding sequences operably linked to a promoter not natively associated with the coding sequence. The coding sequences encode Pichia stipitis proteins that allow recombinant strains of Saccharomyces cerevisiae expressing the protein to grow on xylose, and allow or increase uptake of xylose by Pichia stipitis or Saccharomyces cerevisiae expressing the coding sequences. Expression of the coding sequences enhances uptake of xylose and/or glucose, allowing increased ethanol or xylitol production.
Core Innovation
The invention provides nucleic acid constructs comprising coding sequences operably linked to promoters not natively associated with the coding sequences. These coding sequences encode Pichia stipitis proteins, specifically glucose/xylose transporter polypeptides with at least 95% amino acid identity to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, which when expressed in recombinant Saccharomyces cerevisiae strains enable growth on xylose and enhance uptake of xylose and glucose. Expression of these coding sequences in yeast strains improves sugar uptake, facilitating increased ethanol or xylitol production from xylose-containing media.
The invention addresses the challenge of efficient fermentation of xylose to ethanol by yeast, especially Saccharomyces cerevisiae, which naturally cannot grow on or ferment xylose. While Pichia stipitis ferments xylose, its rate and yield are low compared to glucose fermentation. The problem involves improving sugar uptake, particularly xylose transport, to enhance fermentation rates and yields. Existing transporters have low affinity or specificity for xylose, and the presence of glucose inhibits xylose uptake until glucose is consumed, limiting ethanol production efficiency.
The invention identifies and characterizes novel sugar transporter coding sequences from Pichia stipitis, including XUT1 and SUT4, that have increased expression under xylose growth conditions and have improved affinities and capacities for xylose uptake. Expression of these transporters in Saccharomyces cerevisiae and Pichia stipitis is demonstrated to increase xylose uptake rates, growth on xylose, and production of ethanol and xylitol. The nucleic acid constructs can utilize constitutive or inducible promoters such as the Pichia stipitis fatty acid synthase 2 promoter, especially under oxygen-limiting conditions favorable to fermentation.
Claims Coverage
The patent claims include one independent nucleic acid construct claim and one independent yeast strain claim, covering nucleic acid constructs encoding glucose/xylose transporters, their expression systems, and the resulting yeast strains with enhanced traits.
Nucleic acid construct encoding a glucose/xylose transporter
A nucleic acid construct comprising a first coding sequence operably linked to a promoter not natively associated with the coding sequence, encoding a glucose/xylose transporter polypeptide having at least 95% amino acid identity to SEQ ID NO:2.
Yeast strain comprising nucleic acid construct(s) encoding glucose/xylose transporters
A yeast strain comprising the nucleic acid construct with at least one coding sequence encoding a glucose/xylose transporter polypeptide with at least 95% amino acid identity to SEQ ID NO:2, optionally further comprising a second coding sequence encoding a glucose/xylose transporter polypeptide with at least 95% amino acid identity to SEQ ID NO:4, wherein the strain exhibits increased xylose uptake, higher specific ethanol production rate, and/or increased xylitol production relative to a control yeast lacking the construct, and may have reduced xylitol dehydrogenase activity.
The claims cover nucleic acid constructs encoding Pichia stipitis-derived glucose/xylose transporters operably linked to non-native promoters and yeast strains transformed with these constructs that demonstrate enhanced xylose uptake, increased ethanol production, and improved xylitol production, thereby improving fermentation capabilities.
Stated Advantages
Enables yeast strains, including Saccharomyces cerevisiae, to grow on xylose.
Enhances uptake of xylose and/or glucose by yeast strains.
Increases rates of ethanol and xylitol production from xylose-containing media.
Improves fermentation rates and yields, facilitating more economical large-scale ethanol production from mixed sugar biomass.
Allows for potentially faster and more complete utilization of sugars, including simultaneous uptake of glucose and xylose.
The invention supports yeast growth and fermentation under oxygen-limiting conditions favorable for ethanol production.
Documented Applications
Producing ethanol by fermenting xylose-containing materials using yeast strains expressing the nucleic acid constructs.
Producing xylitol by fermenting xylose-containing materials with yeast strains having reduced xylitol dehydrogenase activity.
Use of engineered yeast strains in fermentation of mixed sugars derived from biomass hydrolysates such as agricultural wastes, wood, paper, and cellulosic materials.
Improving industrial-scale ethanol production processes by employing yeast with enhanced sugar uptake and fermentation capabilities.
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