IP Library Granted Patent US 12,553,066
Granted Patent B2
US 12,553,066 · App. 18/745,084 · Granted Feb 17, 2026

Gene duplications for crabtree-warburg-like aerobic xylose fermentation

Inventors: Chris Todd Hittinger (Madison, WI); Trey Sato (Madison, WI); Sae-Byuk Lee (Madison, WI)
Assignee: Wisconsin Alumni Research Foundation
C12P7/10C10L1/02C12N9/0093C12N9/1022C12N9/1205C12N9/92C12Y117/99C12Y202/01002C12Y207/01017C12Y503/01005C10L2200/0469
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Quick Facts
Patent No.
US 12,553,066
App. No.
18/745,084
Granted
Feb 17, 2026
Kind
B2
Abstract

An engineered yeast strain capable of efficient fermentation of xylose to ethanol, and methods of making and using the strain, are provided.

Claims (75)

1 . A recombinant yeast comprising:

one or more recombinant nucleic acids that recombinantly express one or more of:

a xylulokinase;

a xylose isomerase;

a transaldolase; and

a transketolase; and

one or more disabling mutations of or in one or more of:

a Hog1 gene;

an Isu1 gene;

an Ira1 or Ira2 gene; and

a Gre3 gene.

2 . The recombinant yeast of claim 1 , wherein the recombinant yeast comprises one or more disabling mutations of or in one or more of:

a Hog1 gene;

an Isu1 gene; and

an Ira1 or Ira2 gene.

3 . The recombinant yeast of claim 2 , wherein the one or more recombinant nucleic acids recombinantly express each of:

a xylulokinase;

a xylose isomerase; and

a transketolase.

4 . The recombinant yeast of claim 3 , wherein the recombinant yeast comprises one or more disabling mutations of or in each of:

a Hog1 gene;

an Isu1 gene; and

an Ira1 or Ira2 gene.

5 . The recombinant yeast of claim 3 , wherein the recombinant yeast comprises one or more disabling mutations of or in each of:

a Hog1 gene;

an Isu1 gene;

an Ira1 or Ira2 gene; and

a Gre3 gene.

6 . The recombinant yeast of claim 2 , wherein the one or more recombinant nucleic acids recombinantly express each of:

a xylulokinase;

a xylose isomerase;

a transaldolase; and

a transketolase.

7 . The recombinant yeast of claim 6 , wherein the recombinant yeast comprises one or more disabling mutations of or in each of:

a Hog1 gene;

an Isu1 gene; and

an Ira1 or Ira2 gene.

8 . The recombinant yeast of claim 6 , wherein the recombinant yeast comprises one or more disabling mutations of or in each of:

a Hog1 gene;

an Isu1 gene;

an Ira1 or Ira2 gene; and

a Gre3 gene.

9 . The recombinant yeast of claim 1 , wherein the one or more recombinant nucleic acids recombinantly express each of:

a xylulokinase;

a xylose isomerase;

a transaldolase; and

a transketolase.

10 . The recombinant yeast of claim 1 , wherein the one or more recombinant nucleic acids comprise any combination of:

at least two genes that recombinantly express a xylulokinase;

at least two genes that recombinantly express a xylose isomerase; and

at least one gene that recombinantly expresses a transketolase.

11 . The recombinant yeast of claim 1 , wherein the one or more recombinant nucleic acids comprise at least two genes that recombinantly express a xylose isomerase.

12 . The recombinant yeast of claim 11 , wherein the recombinant yeast comprises one or more disabling mutations of or in each of:

a Hog1 gene;

an Isu1 gene; and

an Ira1 or Ira2 gene.

13 . The recombinant yeast of claim 1 , wherein the one or more recombinant nucleic acids comprise each of:

at least two genes that recombinantly express a xylulokinase;

at least two genes that recombinantly express a xylose isomerase;

at least one gene that recombinantly expresses a transaldolase; and

at least one gene that recombinantly expresses a transketolase.

14 . The recombinant yeast of claim 13 , wherein the recombinant yeast comprises one or more disabling mutations of or in each of:

a Hog1 gene;

an Isu1 gene; and

an Ira1 or Ira2 gene.

15 . The recombinant yeast of claim 14 , wherein the recombinant yeast exhibits aerobic xylose fermentation.

16 . The recombinant yeast of claim 13 , wherein the recombinant yeast comprises one or more disabling mutations of or in each of:

a Hog1 gene;

an Isu1 gene;

an Ira1 or Ira2 gene; and

a Gre3 gene.

17 . The recombinant yeast of claim 16 , wherein the recombinant yeast exhibits aerobic xylose fermentation.

18 . The recombinant yeast of claim 1 , wherein the recombinant yeast exhibits aerobic xylose fermentation.

19 . A method of fermenting xylose comprising contacting the xylose with the recombinant yeast of claim 1 to thereby ferment the xylose.

20 . The method of claim 19 , wherein the fermenting the xylose converts the xylose into ethanol.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 19, 2024
From: HITTINGER, CHRIS; SATO, TREY; LEE, SAE-BYUK
To: WISCONSIN ALUMNI RESEARCH FOUNDATION
Reel/Frame 067770/0834 →
Continuity (3)
Continuation 18052410 · Nov 3, 2022
Provisional Application 63275308 · Nov 3, 2021
Related Publication 20250034599A1 · Jan 30, 2025
References Cited (100)
US 4683195A · Mullis · 1987 [cited by applicant]
US 12049660B2 · Hittinger · 2024 [cited by examiner]
WO WO9914335A1 · 1999 [cited by applicant]
WO WO0071738A1 · 2000 [cited by applicant]
WO WO0242471A2 · 2002 [cited by applicant]
WO WO03049525A2 · 2003 [cited by applicant]
WO WO03102152A2 · 2003 [cited by applicant]
WO WO03102201A2 · 2003 [cited by applicant]
B. Perbal, A Practical Guide To Molecular Cloning (1984) (Book). [cited by applicant]
Bamba et al., Disruption of PHO13 improves ethanol production via the xylose isomerase pathway, AMB Express. 6:4 (2016). [cited by applicant]
Barros et al., Involvement of mitochondrial ferredoxin and Cox15p in hydroxylation of heme O, FEBS Lett., 492:133 (2001). [cited by applicant]
Bracher et al., Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved [cited by applicant]
Brat et al., Functional Expression of a Bacterial Xylose Isomerase in [cited by applicant]
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press (Book). [cited by applicant]
Conant & Wolfe, Functional Partitioning of Yeast Co-Expression Networks after Genome Duplication, PLoS Biol., 4:e109 (2006). [cited by applicant]
Crabtree, Observations on the Carbohydrate Metabolism of Tumours, Biochem J., 23:536 (1929). [cited by applicant]
Culture of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987) (Book). [cited by applicant]
Cunha et al., Xylose fermentation efficiency of industrial [cited by applicant]
Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York (1987) (Book). [cited by applicant]
Dashko et al., Why, when, and how did yeast evolve alcoholic fermentation?, Fems. Yeast Res., 14:826 (2014). [cited by applicant]
Demeke et al., Development of a D-xylose fermenting and inhibitor tolerant industrial [cited by applicant]
Demeke et al., Rapid Evolution of Recombinant [cited by applicant]
DNA Cloning, vols. I and II (D. N. Glover ed., 1985) (Book). [cited by applicant]
Dos Santos et al., Unraveling the genetic basis of xylose consumption in engineered [cited by applicant]
Feng et al., Signature pathway expression of xylose utilization in the genetically engineered industrial yeast [cited by applicant]
Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory) (Book). [cited by applicant]
Gietz et al., High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method, Nat. Protoc., 2:31 (2007). [cited by applicant]
Glerum et al., COX15 Codes for a Mitochondrial Protein Essential for the Assembly of Yeast Cytochrome Oxidase, J. Biol. Chem., 272:19088 (1997). [cited by applicant]
Guldener et al., A new efficient gene disruption cassette for repeated use in budding yeast, Nucleic Acids Res., 24:2519 (1996). [cited by applicant]
Hagman & Piskur, A Study on the Fundamental Mechanism and the Evolutionary Driving Forces behind Aerobic Fermentation in Yeast, PLoS One, 10:e0116942 (2015). [cited by applicant]
Hahn-Hagerdal et al., Towards industrial pentose-fermenting yeast strains, Appl. Microbiol. Biotechnol., 74:937 (2007). [cited by applicant]
Handbook of Experimental Immunology, vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986 (Book). [cited by applicant]
Higgins et al., Natural Variation in the Multidrug Efflux Pump SGE1 Underlies Ionic Liquid Tolerance in Yeast, Genetics, 210:219 (2018). [cited by applicant]
Hittinger & Carroll, Gene duplication and the adaptive evolution of a classic genetic switch, Nature, 449:677 (2007). [cited by applicant]
Huberts et al., A flux-sensing mechanism could regulate the switch between respiration and fermentation, Fems. Yeast Res., 12:118 (2012). [cited by applicant]
Immobilized Cells And Enzymes (IRL Press, 1986) (Book). [cited by applicant]
Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987) (Book). [cited by applicant]
Jeong et al., Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in [cited by applicant]
Jin et al., [cited by applicant]
Jin et al., Conversion of Xylose to Ethanol by Recombinant [cited by applicant]
Johansson et al., Xylulokinase Overexpression in Two Strains of [cited by applicant]
Johnston & Kim, Glucose as a hormone: receptor-mediated glucose sensing in the yeast [cited by applicant]
Kayikci & Nielsen, Glucose repression in [cited by applicant]
Kim et al., Strain engineering of [cited by applicant]
Koppram et al., Evolutionary engineering strategies to enhance tolerance of xylose utilizing recombinant yeast to inhibitors derived from spruce biomass, Biotechnol. Biofuels 5:32 (2012). [cited by applicant]
Kotter & Ciriacy, Xylose Fermentation by [cited by applicant]
Kuyper et al., Metabolic engineering of a xylose-isomerase-expressing [cited by applicant]
Kwak & Jin, Production of fuels and chemicals from xylose by engineered [cited by applicant]
Landi et al., Effect of auxotrophies on yeast performance in aerated fed-batch reactor, Biochem. Biophys. Res. Commun., 414:604 (2011). [cited by applicant]
Langdon et al., sppIDer: A Species Identification Tool to Investigate Hybrid Genomes with High-Throughput Sequencing, Mol. Biol. Evol., 35:2835 (2018). [cited by applicant]
Lau & Dale, Cellulosic ethanol production from AFEX-treated corn stover using [cited by applicant]
Lee et al., Directed Evolution of Xylose Isomerase for Improved Xylose Catabolismand Fermentation in the Yeast [cited by applicant]
Lee et al., Structure-based directed evolution improves [cited by applicant]
Lee et al., Systematic and evolutionary engineering of a xylose isomerase-based pathway in [cited by applicant]
Lee et al., Crabtree/Warburg-like aerobic xylose fermentation by engineered [cited by applicant]
Li, Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM, ar Xiv preprint arXiv: 1303.3997 (2013). [cited by applicant]
Lin & Li, Expansion of Hexose Transporter Genes Was Associated with the Evolution of Aerobic Fermentation in Yeasts, Mol. Biol. Evol., 28:131 (2011). [cited by applicant]
Litsios et al., Metabolic-flux dependent regulation of microbial physiology, Curr. Opin. Microbiol., 42:71 (2018). [cited by applicant]
Lynd et al., Microbial Cellulose Utilization: Fundamentals and Biotechnology, Microbiol. Mol. Biol. Rev. 66:506-577 (2002). [cited by applicant]
Magtanong et al., Dosage suppression genetic interaction networks enhance functional wiring diagrams of the cell, Nat. Biotechnol., 29:505 (2011). [cited by applicant]
McIlwain et al., Genome Sequence and Analysis of a Stress-Tolerant, Wild-Derived Strain of [cited by applicant]
McKenna et al., The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data, Genome Res., 20:1297 (2010). [cited by applicant]
Methods in Enzymology (Academic Press, Inc., N.Y.) (Book). [cited by applicant]
Methods in Enzymology, vols. 154 and 155 (Wu et al. eds.) (Book). [cited by applicant]
Molecular Cloning a Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) (Book). [cited by applicant]
Myers et al., Rewired cellular signaling coordinates sugar and hypoxic responses for anaerobic xylose fermentation in yeast, PLoS Genet., 15:e1008037 (2019). [cited by applicant]
Narayanaswamy et al., Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production, Bioresour. Technol., 102:6995 (2011). [cited by applicant]
Ni et al., Transposon Mutagenesis to Improve the Growth of Recombinant [cited by applicant]
Niebel et al., An upper limit on Gibbs energy dissipation governs cellular metabolism, Nat. Metab., 1:125 (2019). [cited by applicant]
Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984) (Book). [cited by applicant]
Oligonucleotide Synthesis (M. J. Gait ed., 1984) (Book). [cited by applicant]
Osiro et al., Assessing the effect of D-xylose on the sugar signaling pathways of [cited by applicant]
Osiro et al., Exploring the xylose paradox in [cited by applicant]
Parreiras et al., Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant [cited by applicant]
Pauly & Keegstra, Cell-wall carbohydrates and their modification as a resource for biofuels, Plant J., 54:559 (2008). [cited by applicant]
Pfeiffer & Morley, An evolutionary perspective on the Crabtree effect, Front. Mol. Biosci., 1:17 (2014). [cited by applicant]
Pronk et al., Pyruvate Metabolism in [cited by applicant]
Reider Apel et al., Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in [cited by applicant]
Runquist et al., Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing [cited by applicant]
Salusjarvi et al., Regulation of xylose metabolism in recombinant [cited by applicant]
Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual (3rd edition), (Book). [cited by applicant]
Sato et al., Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by [cited by applicant]
Scalcinati et al., Evolutionary engineering of [cited by applicant]
Schwalbach et al., Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an [cited by applicant]
Sherman, Getting Started with Yeast, Methods Enzymol., 350:3 (2002). [cited by applicant]
Stoneman et al., CRISpy-Pop: A Web Tool for Designing CRISPR/Cas9-Driven Genetic Modifications in Diverse Populations, G3 (Bethesda), 10:4287 (2020). [cited by applicant]
Sun & Jin, Xylose assimilation for the efficient production of biofuels and chemicals by engineered [cited by applicant]
Tanaka et al., IRA1, an Inhibitory Regulator of the RAS-Cyclic AMP Pathway in [cited by applicant]
Thompson et al., Evolutionary principles of modular gene regulation in yeasts, eLife, 2:e00603 (2013). [cited by applicant]
Traff et al., Deletion of the GRE3 Aldose Reductase Gene and Its Influence on Xylose Metabolism in Recombinant Strains of [cited by applicant]
Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984) (Book). [cited by applicant]
Verhoeven et al., Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered [cited by applicant]
Wagner et al., PKA and HOG signaling contribute separable roles to anaerobic xylose fermentation in yeast engineered for biofuel production, PLoS One, 14:e0212389 (2019). [cited by applicant]
Walfridsson et al., Xylose-Metabolizing [cited by applicant]
Warburg et al., The Metabolism of Tumors in the Body, J. Gen. Physiol., 8:519 (1927). [cited by applicant]
Williams et al., Integration of Pretreatment With Simultaneous Counter-Current Extraction of Energy Sorghum for High-Titer Mixed Sugar Production, Front Energy Res., 6: (2019). [cited by applicant]
Xu, Haiqing, “PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomy cescerevisiae”, Science Direct Metabolic Engineering 34, (2016), 9 pg… [cited by applicant]
Yamanaka, Inhibition of D-xylose isomerase by pentitols and D-lyxose, Arch. Biochem. Biophys., 131:502 (1969). [cited by applicant]
Zeng et al., Comparative transcriptomes reveal novel evolutionary strategies adopted by [cited by applicant]
Zhang et al., Production of hydrolysates from unmilled AFEX-pretreated switchgrass and comparative fermentation with Zymomonas mobilis, Bioresource Tech. Rep., 11:100517 (2020). [cited by applicant]