USOO8105811 B2 (12) United States Patent (10) Patent No.: US 8,105,811 B2 Jeffries et al. (45) Date of Patent: Jan. 31, 2012 (54) SUGAR TRANSPORTSEQUENCES, YEAST OTHER PUBLICATIONS STRANS HAVING IMPROVED SUGAR Nature Biotechnology 25: 319-326 (Mar. 4, 2007, published on UPTAKE, AND METHODS OF USE line).* Nature Biotechnology 25: 319-326 (Mar. 4, 2007, published on (75) Inventors: Thomas William Jeffries, Madison, WI line)—Supplementary Fig.1. (US); JuYun Bae, Madison, WI (US); With by i. al., St. and charactic "G Bernice Chin-yun Lin, Cupertino, CA E.CS Molecular- SECOC9 SUCOS 1999,aSOOCS s OleF. WeaS Blackwell Cla (US); Jennifer Rebecca Headman Van Science Ltd, Dusseldorf, Germany. Vleet, Visalen, CA (US) Hamacher, Tanja, et al., Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose (73) Assignees: Wisconsin Alumni Research utilization, Microbiology, 2002, 2783-2788, 148, SGM, Great Brit Foundation, Madison, WI (US); The ain. United States of America as R . instNR P3 st site R. 2. 19 represented by the Secretary of Wirginipotis,retrieved Aug. 25, Vailable on thein Internet:=65. : p : Agriculture, Washington, DC (US) Jeffries, Thomas W. et al., “Pichia stipitis genomics, transcriptomics, (*) Notice: Subject to any disclaimer, the term of this Jeffries,and gene Thomas clusters'; W. 2009, et al.; FEMSYeast UniProtKB/TrEMBL, Res., vol.9, No. A3GIFO 6,pp. 793-807. online patent is extended or adjusted under 35 Mar. 2, 2010 (retrieved Aug. 25, 2011). Available on the internet: U.S.C. 154(b) by 235 days. <URL: http://www.uniprot.org/uniprot/A3GIFO.txt?version=24. Jeffries, Thomas W. et al.; UniProtKB/TrEMBL. A3MOD0 online (21) Appl. No.: 12/484,714 Mar. 2, 2010 (retrieved Aug. 25, 2011). Available on the internet: <URL: http://www.uniprot.org/uniprot/A3MOD0.txt?version=28. (22) Filed: Jun. 15, 2009 Jeffries, Thomas W. et al.; UniProtKB/TrEMBL. A3GF74 online Mar. 2, 2010 (retrieved Aug. 25, 2011). Available on the internet: O O <URL: http://www.uniprot.org/uniprot/A3GF74.txt?version=22. (65) Prior Publication Data Koetter y E. online Mar. 2, 2010 US 2009/0325241 A1 Dec. 31, 2009 (retrieved Aug. 25, 2011). Available on the internet: <URL: http:// www.uniprot.org/uniprot/P22144.txt?version=69. Related U.S. Application Data * cited by examiner (60) Provisional application No. 61/061,417, filed on Jun. 13, 2008. Primary Examiner — Tekchand Saidha (74) Attorney, Agent, or Firm — Kilpatrick Townsend & (51) Int. Cl. Stockton LLP CI2P 7/06 (2006.01) CI2N L/4 (2006.01) (57) ABSTRACT CI2N IS/00 (2006.01) Disclosed are nucleic acid constructs comprising coding C7H 2L/02 (2006.01) sequences operably linked to a promoter not natively associ (52) U.S. Cl. .................... 435/161; 435/69. 1; 435/320.1; ated with the coding sequence. The coding sequences encode 435/255.1; 435/255.5; 536/23.1 Pichia stipitis proteins that allow recombinant strains of Sac (58) Field of Classification Search .................. 435/105, charomyces cerevisiae expressing the protein tO grow on 435/252.3,320.1, 161,255.1, 255.5; 536/23.1 xylose, and allow or increase uptake of xylose by Pichia See application file for complete search history. stipitis or Saccharomyces cerevisiae expressing the coding sequences. Expression of the coding sequences enhances (56) References Cited uptake of xylose and/or glucose, allowing increased ethanol or xylitol production. U.S. PATENT DOCUMENTS 4,701.414 A 10/1987 Van Dijken et al. 19 Claims, 8 Drawing Sheets U.S. Patent Jan. 31, 2012 Sheet 1 of 8 US 8,105,811 B2 ClustalW (v1. 4) multiple sequence alignment 3 Sequences Aligned Alignment Score = 34227 Gaps Inserted = 0 Conserved Identities = 1606 Pairwise Alignment Mode: Slow Pairwise Alignment Parameters: Open Gap Penalty = 10. O Extend Gap Penalty = 0.1 Multiple Alignment Parameters: Open Gap Penalty = 10.0 Extend Gap Penalty = 0.1 Delay Divergent = 40% Transitions: Weighted Processing time: 3.3 seconds SUT4 ORE 1 ATGTCCTCACAAGATTTACCCTCGGGTGCTCAAACCCCAATCGATGGC SO (SEQ ID NO : 3) SUT2nt mvr ATGTCCTCACAAGATTTACCCTCGGGTGCTCAAACCCCAATCGATGGTTC SO (SEQ ID NO: 12) slut3 AGTCCTCACAAGATTACCCTCGGGTGCTCAAACCCCAA CGATGGTC SO (SEQ ID NO: 13) k . SUT4 ORF 51 TTCCACCCGAAGAAAAGTTGAGCAAAGTTCGTCCCAAATAGCCAAA OO SUT2nt. nV 51 CCATCCTCGAAGATAAAGTTGAGCAAAGTTCGTCCT CAAATAGCCAAA OO Sut 3 51 CCATCCTCGAAGATAAAGTTGAGCAAAGTTCGTCCT CAAATAGCCAAA OO k . SUT4 ORF O 1 GTGATTTAGCTTCIATTCCAGCAACAGGTACAAAGCCTATCTCTTGGTT SO SUT2nt my 101 GTGATTTAGCTTCCATTCCAGCAACAGGTATCAAAGCCTATCTCTTGG 15 O Sut 3 O1 GTGATTTAGCTTCGATTCCACCAACAGGTATCAA AGCCTATCTCTTGG 50 ". .33: . SUT4 ORF 151. TGTTTCTTCTGCATGTTGGTTGCCTTCGGTGGGTTCGTATTCGGTTTCGA 200 S2 ... n. my 151. TGTTTCTTCTGCATGTTGGTTGCCTTEGGTGGATTCGTATTCGGTTTCGA 2 OO Slt3 151 TGTTTCTTCTGCATGTTGGTTGCCTTTGGTGGGTTCGTATTCGGTTTCGA 200 k . k. kšk. k. k: . SUT4 ORF 201 TACCGGTACTATTTCCGGTTTCCTTAATATGTCTGATTTCCTTTCCAGAT 250 SUT2 ... t. wr 2O1 TACCGGTACAATTTCCGGTTTCCTTAATATGTCTGATTTCCTTTCCAGAT 2 SO slut3 2O1 TACCGGTACAATTTCCGGTTTCCTTAATATGTCTGATTTCCTTTCCAGAT 25 O k kirk k ki krikk ke k l k k l k kk. k.k. k.k. k.k. k. k.k. k. k. k.k. k. k.k. k. k. k.k. k.k kk. k.k. k. SUT4 ORF 251 GGTCAAGATGGTTCTGAAGGAAAATATTTGTCTGATATCAGAGTCGG 3 OO SUT2. Ilt. Inv 251 TTGGTCAAGATGGTTCTGAAGGAAAATATTTGTCTGATATCAGAGTCGG 3 OO Sut3 251 TTGGTCAAGATGGTTCTGAAGGAAAATATTTGTCTGATATCAGAGTCGG 3 OO k . SUT4 ORF 3 O 1 TTGATTGTTTCCATTTTTAACATTGGTTGTGCAATTGGTGGTATTTTCC 35 O SUT2nt. nV 3 O1 GATTGTTTCCA TTAACATTGGTTGTGCAATTGGTGGTA ICC 350 slut 3 3 O 1 TTGATTGTTTCCA TTAACATTGGTTGTGCAATTGGTGGTATTTTCC 350 . SUT4 ORE 35 TTCTAAGATAGGAGAGTTTACGG TAGAAGAATTGGTATCATTCAGOA. A OO SUT2 intmvr 35 TTCTAAGATAGGAGAGTTTACGG TAGAAGAATTGGTATCATTCAGOA. A OO Sut 3 35 TTCTAAGATAGGAGATGTTTACGGTAGAAGAATTGGTATCATTCAGOA 4 OO . SUT4 ORF 401 TGGTTGTCTATGTCGTCGGTATAATCATCCAGATCTCGTCCCAAGAAAG 450 SUT2 ... it mvr 401 TGGTTGTCTACGTCGTCGGTATTATCATCCAGATCTCGTCCCAAGACAAG 450 suit 3 401 TGGTTGTATACGTCGTCGGTATTATCATCCAGATCTCGTCCCAAGACAAG 450 kk k k + k ki k k sk k + k l k k + k l k + k + k k k l k l k k + k l k k.k k + k l k kick k SUT4 ORF As TGGTACAACTTACAATTGGACGTGGAGTTACAGGATTAGCTGTTGGTAC 5 OO SUT2 ... t. IV 451. TGGTACCAACTTACAATTGGACGGGAGTTACAGGATTAGCGTTGGTACFIG. 1 (1 of 3) 5 OO U.S. Patent Jan. 31, 2012 Sheet 2 of 8 US 8,105,811 B2 Sut 3 451. TGGTACAACTTACAATTGGACGTGGAGTACAGGATTAGCGTGGTAC 5 OO k k.k k k ... k k k k k is k k k k k k k l k k.k. k. k. k.k. k.k. k. k.k. k. k. k. k.k. k.k. k. k. k.k kk. k. k. k.k SUT4 ORF 501 TGTTTCGGTTTTGTCTCCAATGTTCATTAGTGAAAGTGCTCCAAGCATT 550 SUT2 ...tv 501 TGTTTCAGTGTTGTCTCCAATGTTCATTAGTGAAAGTGCTCCAAAGCATT 550 Sut 3 5 O1. TGTTTCAGTGTTGTCTCCAATGTTCATTAGTGAAAGTGCTCCAAAGCATT 550 :: * : . : SUT4 ORF 551. TGAGAGGTACTTTGGTATACTGTTACCAATTATGEATCACCTTAGGTATT 6 OO SUT2 ... nt mV. E. 51. TGAGAGGTACTTTGGTATACTGTTACCAATTATGEATCACCTTAGGTATT 6 OO slut 3 SS1 TGAGAGGTACTTTCCTATACTGTTACCAATTATGTATCACCTTAGGTATT 6 OO k . ex: k . SUT4 ORF 6O1 CATGGTACTGTGTCACTATGGAACCAA AGATTTAAATGATTCAAG 65 O SUT2 int. nV 6 O TTCATTGGTTACTGGTCACTTATGGAACCAAAGATTTAAATGATTCAAG 65 O Sut3 6 O TCATTGGTTACTGGTCACTTATGGAACCAAAGATTAAATGATCAAG 650 k . k . SUT4 ORF 651 ACAATGGAGAGTTCCTTTGGGTTTATGTTTCCTTTGGGCTATTTTCTTAG 700 SUT2 intmvr 651 ACAATGGAGAGTTCCTTTGGGTTTATGTTTCCTTTGGGCTATTTTCTTAG 700 Sut 3 651 ACAATGGAGAGTTCCTTTGGGTTATGTTCCTTTGGGCTA CTTAG 7 OO sk k k k k l k k k k k k k k k k + k k k k . k + k k k.k. k. k. k.k. k. k. k. k. k. k. k. k. k. k. k. k. k.k. k. k. k. SUT4 ORF 701 TTGTGGTATGTTGGCTATGCCAGAATCCCCAAGATTCTTGATTGAAAAG 750 SUT2 ... nt inv 701 TTGTCGGTATGTTGGCTATGCCTGAATCCCCAAGATTCTTAATTGAAAAG 750 Sut 3 701 TTGTCGGTATGTTGGCTATGCCAGAATCCCCAAGATTCTTAATTGAAAAG 750 k k: ; k ki ki k ki ke k k is k is k Ek k k k k k k k k ki: ; k ki k k SUT4 ORF 751. AAGAGAATCGAAGAAGCCAAGAAGTCCCTTGCAAGATCCAACAAGTTGTC 8 OO SUT2 intmvr 75 AAGAGAATCGAAGAAGCCAAGAAGTCCCTTG-CAAGACCAACAAGTTATC 8 OO sult 3 75 AAGAGAATCGAAGAAGCCAAGAAGTCCCTTGCAAGATCCAACAAGTTATC 8 OO k k + k k k + k k + k k k k k k k k k k l k k k k k k k k k kk. k.k k k k k k k k k k k k + k SUT4 ORF 801 TCCAGAAGATCCAGGTGTCTACACTGAA CAATTGATTCAAGCTGGTA 850 SUT2 ...tv 801 TCCAGAAGATCCAGGTGTCTACACTGAA CAATTGATTCAGGCTGGTA 8 SO Sut 3 8 O TCCGAAGATCCAGGTGTCTACACTGAA CAATTGATTCACGCTGGTA 85 O k k . k is k.k k . k k k l k . k h k l k sk k . k 3: SUT4 ORF 851 TTGACAGAGAAGCTGCTGCAGGTTCTGCTTCGTGGATGGAATTGATTACC 900 SUT2 ..lt. nV 851 TTGACAGAGAAGCTGCTGCAGGTTCTGCTTCATGGATGGAATTGATCACT 900 Slut 3 851 TTGACAGAGAAGCTGCTGCAGGTTCTGCTTCGTGGATGGAATGATO ACT 9 OO sk k + k k k k k k + k k k + k k + k k + k k k + k k kk. k. k. k.k. k.k. k. k.k. k.k k + k k k kix k SUT4 ORF 901 GGTAAACCAGCTATTTTCAGAAGAGTTATCATGGGAATTATCTTACAGTC 950 SUT2 intmvr 901 GGTAAGCCAGCTATTTTCAGAAGAGTTATCATGGGAATTATCTTACAGTC 950 Sut3 9 O. GGTAAGCCAGCTATTTTCAGAAGAGTTATCATGGGAATTATCTTCAGTC 950 : . SUT4 ORF 951 TTTGCAGCAATTAACTGGTGTCAACTATTTCTTCTACTACGGAACTACAA 1000 SUT2 ... lt. nV 951 TTTGCAACAATTAACTGGTGTCAACTATTTCTTCTATTACGGAACTACAA 1000 Sut 3 951 TTTG-CAACAATTAACTGGTGTCAACTATTTCTTCTATTACGGAACTACAA 10 OO : . : . SUT4 ORF OOL TCTTCCAAGCTGTIGGTTTGCAAGATTCCTTCCAGACTTCCATCATCTTA, OSO SUT2nt nv 1 OO1 TCTTCCAAGCTGTTGGTTTGCAAGATTCCTTCCAGACTTCCATCATCTTA 1 O5 O Sut 3 OO, TCTCCAAGCTGITGGTTTGCAAGATTCCTTCCAGACTCCATCATCTA 1 OSO k . k . SUT4 ORF O 51 GGTACAGTCAACTTCTTTCTACATTTGTTGGTATTTGGGCCATTGAAAG 11 OO SUT2 . It nv OS, GGTACAGICAACTITCTTTCACATTTGITGGTATTIGGGCCATIGAAAG 11 OO Sut 3 O5, GGTACAGTCAACTTTCTTTCACATTTGTTGGTATTGGGCCATTGAAAG OO k . SUT4 ORF O 1 ATTTGGAAGAAGACAATGTTTCTTAGTCGGTTCTGCTGGTAGTTCGTTT 15 O SUT 2 int. myr 11 O ATTTGGAAGAAGACAATGTTTGTTAGTCGGTTCTGCTGGTATGTTCGTTT 115 O slut 3 O ATITGGAAGAAGACAATGTTTGITAGTCGGICIGCTGGIAGITCGTIT