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-8916367-B2
Publication Date
2014-12-23
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 sequence. The coding sequences encode Pichia stipitis proteins, such as 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. Expression of these coding sequences in recombinant yeast strains like Saccharomyces cerevisiae enables growth on xylose and increases uptake of xylose, thereby enhancing ethanol or xylitol production from xylose-containing media.
The problem being solved is the economic and technical challenge of efficiently fermenting xylose from biomass-derived sugars to produce ethanol as an alternative energy source. Saccharomyces cerevisiae, widely used industrially, cannot effectively transport or ferment xylose. Although native xylose-fermenting yeasts like Pichia stipitis can ferment xylose, their fermentation rates and ethanol yields are limited. Moreover, bacteria capable of fermenting pentoses have drawbacks such as sensitivity to low pH and ethanol, as well as low ethanol yields. Therefore, there is a need for yeast strains with improved sugar uptake, especially xylose, to increase fermentation efficiency and ethanol yields from renewable biomass feedstocks.
The invention identifies and utilizes putative sugar transporter genes from Pichia stipitis, including XUT1 and SUT4, which show increased expression in xylose-containing media. Expression of these transporters in S. cerevisiae improves growth on xylose and enhances uptake of xylose and glucose. Variants and polypeptides with amino acid identities above 80%, preferably above 95%, to these sequences are considered within the scope of the invention, with particular attention to sequences having a threonine or serine residue at a key position corresponding to amino acid 544 of SEQ ID NO:4. The invention also contemplates expression of multiple transporter genes in yeast strains to further enhance fermentation performance.
Claims Coverage
The patent includes one independent claim that covers a method involving genetically engineered yeast strains and their use in fermentation processes. The main inventive features relate to the coding sequences and the yeast strains for enhanced sugar uptake and fermentation.
Method of producing ethanol or xylitol using engineered yeast strains
A method comprising culturing a yeast strain that contains a nucleic acid comprising a coding sequence operably linked to a promoter not natively associated with the coding sequence, the coding sequence encoding a glucose/xylose transporter polypeptide with at least 95% amino acid identity to SEQ ID NO:2 or SEQ ID NO:4, wherein the polypeptide expressed includes a threonine residue at the amino acid position corresponding to position 544 of SEQ ID NO:4.
Enhanced xylose uptake in yeast strains
The yeast strain exhibits increased xylose uptake relative to a control yeast lacking the nucleic acid, enabling fermentation of xylose-containing materials to ethanol or xylitol, including in the presence of glucose.
Promoter specificity in nucleic acid constructs
The promoter operably linked to the coding sequence is a constitutive promoter or an inducible promoter inducible under oxygen limiting conditions, such as the FAS2 promoter.
Applicability to yeast species
The yeast strains employed in the method are Saccharomyces cerevisiae or Pichia stipitis, genetically engineered to express the polypeptides, enabling improved fermentation.
Xylitol accumulation through enzymatic activity modulation
Yeast strains may have reduced xylitol dehydrogenase activity to accumulate xylitol during fermentation, resulting in increased xylitol production relative to controls.
The claims cover methods of fermenting xylose-containing materials using genetically engineered yeast strains expressing specific glucose/xylose transporter polypeptides with defined sequence identity and promoter control, resulting in improved sugar uptake, ethanol and xylitol production, across multiple yeast species and fermentation conditions.
Stated Advantages
Expression of the identified sugar transporters enhances uptake of xylose and glucose in yeast, improving growth on xylose and fermentation efficiency.
The engineered yeast strains can ferment xylose more rapidly and produce higher yields of ethanol or xylitol than control strains lacking the transporters.
Expression of SUT4 in Pichia stipitis results in faster glucose and xylose consumption and increases the specific rate of ethanol production by more than threefold compared to control strains.
Use of inducible promoters responsive to oxygen-limiting or xylose-containing conditions enables regulation of sugar transporter expression aligned with fermentation needs.
Documented Applications
Producing ethanol by fermenting xylose-containing materials using genetically engineered yeast strains comprising nucleic acid constructs encoding Pichia stipitis glucose/xylose transporter proteins.
Producing xylitol by fermenting xylose using yeast strains with reduced xylitol dehydrogenase activity to accumulate xylitol.
Fermentation of sugar mixtures derived from biomass hydrolysates such as agricultural wastes, corn hulls, corncobs, cellulosic materials, and pulping wastes containing xylose and glucose.
Using Saccharomyces cerevisiae or Pichia stipitis strains engineered to express transporter polypeptides for improved simultaneous uptake and fermentation of glucose and xylose sugars in industrial biomass conversion processes.
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