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 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.

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 SO FIG. 1 (2 of 3) U.S. Patent Jan. 31, 2012 Sheet 3 of 8 US 8,105,811 B2

k ...... it ......

SUT4 ORF 1151 GTTTCATCATTTACTCTGTATTGGTACAACTCATTTGTTCATTGATGGA 12 OO SUT2.nt.mv 1151 GTTTCATCATTTACTCCGTTATTGGTACAACTCATTTGTTCATTGATGGA 1200 Slut 3 1151 GTTTCATCATTTACTCGGTTATTGGTACAACTCATTTGTTCATTGATGGA 12 OO kkkk k . . k k k k kkkk k.k. k.k k l k kk. k.k. k.k. k.k. k.k kkk k k k k kk. k.k. k. k.k. k.k.

SUT4 ORF 12O1 GTAGTAGATAACGACAACACCCGTCAACTGTCTGGTAATGCTATGATCT 125 O SUT2 ... t.v. 12 O GTAGTAGATAACGACAACACCCGTCAACTGTCTGGTAATGCTATGATCTT 25 O slut3 12 O1 GTAGAGATAACGACAACACCCGTCAACTGTCTGGTAATGCTATGACTT 125 O k ...... k ......

SUT4 ORF 1251 TATCACTTGTTTGTTCATCTTCTTCTTTGCCTGTACTTGGGCTGGAGGTG 1300 SUT 21ht myr 1251 TATCACTTGTTTGTTCATCT TOTTCTTTGCCTGTACATGGGCTGGAGGTG 13 OO Sut3 1251 TATCACTTGTTTGTTCATCTTCTTCTTTGCCTGTACATGGGCTGGAGGTG 13 OO k ...... k ...... SUT4 ORF 1301 TTTTTACAATCATTTCCGAATCATATCCATTGAGAATCAGATCCAAGGCT 1350 SU2 intmw 13 O TTTTACEATCATTTCCGAATCATATCCATTGAGAATCAGATCCAAGGCA 135 O Sut3 13 O1 TTTACCATCA CCGAATCATATCCATTGAGAATCAGATCCAAGGCA 135 O : ......

SUT4 ORF 1351 ATGTCTATTGCCACTGCCGCTAACTGGATGTGGGGTTTCTTGATTTCATT 1400 SUT2 .nt. my 1351 ATGTCTATTGCTACTGCIGCTAACTGGATGTGGGGGTTCTTGATTTCCTT 1400 Sut 3 1351 ATGTCTATTGCTACTGCTGCTAACTGGATGTGGGGGTTCTTGATTTCCTT 1400 sk k k . . . . k is kiškk k kit k k k k . . . . . k. k. k.k. i ki ke k k k k k k k k

SUT4 ORF 1401. CTGCACTCCATTCATTGTTAACGCCATCAACTTCAAGTTCGGCTTTGTGT 1450 SUT2. It mV 14 O1. CTGCACTCCATTCATTGTTAAGCCATCAACTTCAAGTTCGGCTTTGTGT 145 O sut3 14 O1. CTGCACTCCATTCATTGT TAAGCCATCAACTTCAAGTTCGGCTTTGTGT 14 SO k ......

SU4 ORF 1451 TTACTGGTTGT GCTCTTT CGTTCTTCTATGTCTACTTCTTTGTCAGC 15 OO SUT2 intmv 1451 TTACTGGTTGTTTACTCTTTTCGTTCTTCTATGTCTACTTC GTCAGC 15 OO Sult3 14 S1, TTACTGGTTGITTACTCTTTTCGITCTTCTATGTCTACTTCTTTGTCAGC 1. SOO k ...... ki:k k ...... k k k k k

SU4 ORF 15O1 GAAACCAAAGGTTTGTCGTTGGAAGAAGTTGATGAGTTGTACGCTGAGGG 1550 SUT2 ... int. mV l5O1 GAAACCAAAGGTTTGTCGTTGGAAGAAGTTGATGAGTTGTACGCTGAAGG 1550 Sut3 15 O GAAACCAAAGGTTTGTCGTTGGAAGAAGTTGATGAGTTGTACGCTGAAGG 155 O k ...... k 3:

SUT4 ORF 1551 AATTGCACCATGGAAATCGGGTGCATGGGTTCCTCCTTCTGCACAACAAC 1600 SUT2 int. my 1551 TATTGCACCATGGAAGTCGGGCATGGGTTCCTCCTTCTGCCCAACAAC 16 OO Sut3 1551 TATTGCACCATGGAAGTCGGTGCATGGGTTCCTCCTTCTGCCCAACAAC 16 OO sk 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. k. k.k. k.

SUT4 ORF 1601 AAATGCAAAACTCTACTTATGGTGCCG AAGAGCAAGAGCAAGTT 1650 SUT2 intmv 1601 AAATGCAAAACTCCACTTATGGTGCCGAA GAGCAAGAGCAAGT 1650 suits 16O1 AAAIGCAA AACT COACTTATGGTGCCG GAGCAAGAGCAAGTT 1650 k ...... k : k ...... k k k k k k k k

SUT4 ORF 1651 TAG 1653 + + + -- 34 NT changes SUT2 ... t.v 1651 TAG 1653 + + 3 NT changes Sut 3 1651 TAGSTT 1656 ------6 NT changes FIG. 1 (3 of 3) U.S. Patent Jan. 31, 2012 Sheet 4 of 8 US 8,105,811 B2

Growth in xylose

3.O

2.5 -

2.0 -

1.5 -

1.0 -

0.5 -

O.O -

I I 2 Day 4 Day 6 Day 8 Day 10 Day

-O- 314+316 -O- GX316 -V- GXXUT1 partial - V - GXXUT 1 -- GXSUT4

FIG. 2 U.S. Patent Jan. 31, 2012 Sheet 5 of 8 US 8,105,811 B2

Growth in glucose

4

3 -

2 - o 3 ?h O 1 -

O -

-O- 314+316 -O- GX123+316 -v- Gx123+XUT1 partial - V - GX 123+XUT 1 -- GX123+SUT4

FIG. 3 U.S. Patent Jan. 31, 2012 Sheet 6 of 8 US 8,105,811 B2

50 mM 'C-glucose or xylose uptake with time course.

ZOOOO

35OOO

3OOOO

25OOO aga XUT1-G as a SUT4- G 2OOOO & XU1-X

SUTA-X SOOO

OOOO

OOO

O SO 1 OO 1 SO OOO 2SO 3OO 3SO Time (Sec)

FIG. 4 U.S. Patent Jan. 31, 2012 Sheet 7 of 8 US 8,105,811 B2

SUT4 VS Control

8

2 O 1

O 2O 40 60 80

FIG. 5 U.S. Patent Jan. 31, 2012 Sheet 8 of 8 US 8,105,811 B2

SUT4 VS Control

- 30

- 25 | 20 O) 9. CDf - 15 O 5 - 10 - 5

- O O 2O 40 6O 8O Time (Hours) -E-SUT4. Glucose -- Control Glucose ASUT4 Ethanol-A-Control Ethanol

FIG. 6 US 8,105,811 B2 1. 2 SUGAR TRANSPORTSEQUENCES, YEAST In order to improve the cost effectiveness of xylose fermen STRANS HAVING IMPROVED SUGAR tation, it is necessary to increase the rate of fermentation and UPTAKE, AND METHODS OF USE the ethanol yields obtained. The most commonly used yeast in industrial applications is CROSS-REFERENCE TO RELATED Saccharomyces cerevisiae. Although S. cerevisiae is unable APPLICATIONS to grow on or ferment Xylose, homogenates of S. cerevisiae can readily ferment D-ribulose-5-phosphate to ethanol, and This application claims the priority to U.S. Provisional can convert D-xylulose-5-phosphate to a lesser extent. S. Patent Application No. 61/061.417 filed Jun. 13, 2008, which cerevisiae metabolically engineered to overproduce D-xylose is incorporated by reference herein. 10 reductase (XYL1), xylitol dehydrogenase (XYL2) and D-xy lulokinase (XYL3 or XKS1) or some forms of xylose STATEMENT REGARDING FEDERALLY isomerase (xylA) along with AYL3 or XKS1 can metabolize SPONSORED RESEARCH ORDEVELOPMENT Xylose to ethanol. Pichia stipitis is a yeast species that in its native state is able This invention is jointly owned between WARF and the 15 to ferment xylose to produce ethanol. In P stipitis, fermenta USDA and was made with United States government support tive and respirative metabolism co-exist to support cell awarded by the following agencies: growth and the conversion of Sugar to ethanol. USDA Grant Number 2006-35504-17436. There is a need in the art for yeast strains having improved The government has certain rights in this invention. ability to convert Sugars to ethanol. BRIEF SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION In one aspect, the present invention provides a nucleic acid Within the United States, ongoing res is directed toward construct comprising a coding sequence operably linked to a developing alternative energy sources to reduce our depen 25 promoter not natively associated with the coding sequence. dence on foreign oil and nonrenewable energy. The use of The coding sequence encodes a polypeptide having at least ethanol as a fuel has become increasingly prevalent in recent 95% amino acid identity to SEQID NO:2, SEQID NO:4, or years. Currently, corn is the primary carbon source used in SEQID NO:6. ethanol production. The use of corn in ethanol production is Other aspects of the invention provide yeast strains com not economically Sustainable and, arguably, may result in 30 prising the nucleic acid construct, and methods of producing increased food costs. ethanol or Xylitol by growing a yeast strain of the invention in In order to meet the increased demand for ethanol, it will be xylose-containing medium. necessary to ferment Sugars from other biomass, such as agricultural wastes, cornhulls, corncobs, cellulosic materials, BRIEF DESCRIPTION OF THE SEVERAL pulping wastes, fast-growing hardwood species, and the like. 35 VIEWS OF THE DRAWINGS Biomass from most of these sources contains large amounts of xylose, constituting about 20 to 25% of the total dry FIG. 1 shows the alignment of PsSUT2, PsSUT3, and weight. Because agricultural residues have a high Xylose PSSUT4. content, the potential economic and ecologic benefits of con FIG. 2 shows growth, measured as ODoo, of S. cerevisiae Verting Xylose in these renewable materials to ethanol are 40 expressing XUT1 or SUT4 on xylose as a function of time. significant. FIG.3 shows growth, measured as ODoo, of S. cerevisiae In biomass conversion, microorganisms serve as biocata expressing XUT1 or SUT4 on glucose as a function of time. lysts to convert cellulosic materials into usable end products FIG. 4 shows uptake of C-glucose or ''C-xylose, mea Such as ethanol. Efficient biomass conversion in large-scale sured as DPM, by S. cerevisiae expressing XUT1 or SUT4 as industrial applications requires a microorganism that can tol 45 a function of time. erate high Sugar and ethanol concentrations, and which is able FIG.5 shows consumption of glucose and xylose by Pichia to ferment multiple Sugars simultaneously. stipitis overexpressing SUT 4 as a function of time. The pentose D-xylose is significantly more difficult to fer FIG. 6 shows glucose consumption and ethanol production ment than D-glucose. Bacteria can ferment pentoses to etha by Pichia stipitis overexpressing SUT4 as a function of time. nol and other co-products, and bacteria with improved etha 50 nol production from pentose Sugars have been genetically DETAILED DESCRIPTION OF THE INVENTION engineered. However, these bacteria are sensitive to low pH and high concentrations of ethanol, their use in fermentations One approach to increasing the efficiency of yeast-cata is associated with co-product formation, and the level of lyzed conversion of Sugars to ethanol or to other products ethanol produced remains too low to make the use of these 55 Such as Xylitol is to enhance uptake of Sugars by the yeast. bacteria in large-scale ethanol production economically fea Cellulosic and hemicellulosic materials used in fermentation sible. reactions are generally Subjected to pretreatments and enzy In general, industrial producers of ethanol strongly favor matic processes that produce a mixture of several Sugars, using yeast as biocatalysts, because yeast fermentations are including glucose, Xylose, mannose, galactose, and arabi relatively resistant to contamination, are relatively insensitive 60 OS. to low pH and ethanol, and are easier to handle in large-scale Saccharomyces cerevisiae and other hexose-fermenting processing. Many different yeast species use Xylose respira yeasts have Sugar transporters that facilitate uptake of hex tively, but only a few species use xylose fermentatively. Fer oses. Some hexose Sugar transporters also promote uptake of mentation of xylose to ethanol by wild type xylose-ferment pentoses such as Xylose, but at a much lower rate and with ing yeast species occurs slowly and results in low yields, 65 much lower affinity. Therefore, xylose uptake and utilization relative to fermentation rates and ethanol yields that are is impaired until glucose is consumed. When the Substrate obtained with conventional yeasts in glucose fermentations. includes large initial concentrations of glucose, ethanol pro US 8,105,811 B2 3 4 duction from fermentation of glucose may result in inhibitory relative to SEQ ID NO:2 and SEQ ID NO.4 are within the concentrations of ethanol before Xylose uptake begins. Scope of the invention, provided that the polypeptide has at In order to promote Xylose utilization by yeast in fermen least 80% amino acid identity to SEQID NO:2 and SEQID tation of mixed hemicellulosic Sugars, Sugar transport pro NO:4. Suitably, a polypeptide according to the invention has teins having high affinity or specificity for Xylose were iden 5 at least 85%, 90%, or 95% amino acid identity to SEQ ID tified and used to develop yeast engineered to have improved NO:2 or SEQID NO:4. It is further envisioned that polypep xylose uptake, as described below in the Examples. tides according to the invention have at least 80%, 85%, 90%, The genome of the Xylose-fermenting yeast Pichia stipitis 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to was examined for putative Sugar transporters based on simi SEQID NO:4, and have a threonine residue or a serine resi larity to transporters that facilitate uptake of hexoses, lactose, 10 maltose, phosphate, urea, and other compounds. Eight puta due at a position corresponding to amino acid 544 of SEQID tive xylose transporters (XUT1-7 and SUT4) were chosen NO:4. Preferably, polypeptides according to the invention based on their implied structures and sequence similarities to retain their activity, i.e., retain the ability to enhance uptake of other reported Sugar transporters. Genome array expression glucose and/or Xylose. Such activity may be assayed by any studies were used to evaluate whether the Sugar transporters 15 Suitable means, including, for example, those described in the showed induced expression in cells grown on Xylose. examples. Genome array expression results were confirmed using quan It is well understood among those of ordinary skill in the art titative PCR of transcripts from cells grown on xylose. that certain changes in nucleic acid sequence make little or no Sequences encoding polypeptides corresponding to those difference to the overall function of the protein or peptide encoded by two (XUT1 and SUT4) that showed encoded by the sequence. Due to the degeneracy of the increased expression in cells grown on Xylose-containing genetic code, particularly in the third position of codons, media under oxygen limited conditions were expressed in a changes in the nucleic acid sequence may not result in a host strain Saccharomyces cerevisiae JYB3011, which lacks different amino acid being specified by that codon. Changes all hexose-transport genes and expresses P Stipitis XYL1, that result in an amino acid Substitution may have little or no XYL2, and XYL3, which encode xylose reductase (XR), 25 effect on the three dimensional structure or function of the xylitol dehydrogenase (XDH), and xylulokinase (XK), encoded protein or peptide. Conservative Substitutions are respectively. Expression of either XUT1 or SUT4 allowed even less likely to result in a lost of function. In addition, growth of the S. cerevisiae strain on xylose. Both XUT1 and changes that result in insertions or deletions of amino acids SUT4 allowed increased xylose uptake. Cells carrying these may also be acceptable. sequences were tested for their relative affinities in taking up 30 Alignment of the native sequence encoding Pssuta with "C-labeled glucose or "C-labeled xylose. Xut 1 was shown those encoding PsSut2 and PsSut3 shows that PsSut4 differs to have a higher affinity for xylose, and SutA a higher affinity from the coding sequences for PsSut2 and PsSut3 at more for glucose. A transformant carrying a partial sequence of the than 30 bases (FIG. 1), with only two differences resulting in XUT1 gene, which encodes the C-terminal portion of the a change in the amino acid specified. Suitably, polynucle protein, beginning with amino acid residue 154 of SEQ ID 35 otides according to the invention include one or more of the NO:2, was shown to bind xylose and glucose but did not divergent sequences of PsSUT4 shown in FIG. 1, in any accumulate these Sugars, thereby demonstrating that at least combination. Suitably, the polynucleotides include anywhere Some portion of the amino terminus of the protein is needed from one to all of the divergent sequences of PsSUT4 shown for Sugar transport. in FIG. 1. Thus, expression of any of XUT1, XUT1 partial, and SUT4 40 It is expected that expression of more than one of the in Saccharomyces cerevisiae allows growth on Xylose, and in proteins encoded by the polynucleotides of the invention, as the case of XUT1 and SUT4, uptake of xylose. It is envisioned exemplified by SEQ ID NO:2, SEQ ID NO:4, and SEQ ID that expression of any of XUT1, XUT1 partial, and SUT4 or NO:6, could be obtained in a transgenic yeast cell. Expression sequences similar to XUT1, XUT1 partial, and SUT4 in other of two or more polynucleotides of the invention may afford yeast species will also allow growth on Xylose and/or uptake 45 additional advantages in terms of fermentation rate or Sugar of xylose. uptake. For example, one could create a yeast strain that The sequence of the deduced amino acid sequence of the expresses a polynucleotide encoding a polypeptide having at putative proteins encoded by the Pichia stipitis XUT1 and least 80%, at least 85%, at least 90%, or at least 95% amino SUT4 genes are provided in SEQID NO:2 and SEQID NO:4, acid identity to SEQID NO:2 together with a polynucleotide respectively. The sequences of the XUT1 and SUT4 coding 50 encoding a polypeptide having at least 80%, at least 85%, at sequences prepared from Pichia stipitis are shown in SEQID least 90%, or at least 95% amino acid identity to SEQ ID NO:1 and SEQID NO:3, respectively. NO:4. The P stipitis glucose/xylose facilitators PsSut 1-4 have a Additionally, the polynucleotides of the invention may fur high degree of sequence identity, with PsSut1 having 78% ther include a sequence encoding a selectable marker that amino acid identity with PsSut2.3, and 4. PsSut2 differs from 55 would allow selection of yeast expressing the polynucleotide. PsSut3 by one amino acid (L279V). PsSut4 differs from In the examples, expression of SUT4 under the control of PsSut2 by just one amino acid A544T) and from PsSUT3 by the promoter from P. stipitis fatty acid synthase 2 (FAS2 two amino acids (L279V and A544T). While all four facili promoter), which is induced by Xylose and under oxygen tators have similar affinities for glucose, their affinities for limiting conditions, was obtained in S. cerevisiae yJML 123 xylose were shown to differ significantly (See Table 3). Nota 60 and in P stipitis UC7. The yJML 123+SUT4 was able to use bly, PsSut4 has a higher specificity ratio (V/K) than the both glucose and Xylose at a faster rate than the control strain. other Sut facilitators for glucose and Xylose, and a higher Neither strain was able to use all of the xylose. The affinity for xylose than any of the other facilitators. yJML 123+SUT4 strain, which has reduced expression of It is envisioned that changes to the polypeptides could be xylitol reductase, produced a higher yield of xylitol (18.45 made without affecting their activities, and Such variations 65 g/L) than the control strain (15.51 g/L). Further, the are within the scope of the invention. For example, polypep yJML 123+SUT4 strain produced a higher yield of xylitol tides have insertions, deletions or Substitutions of amino acids faster than the control. US 8,105,811 B2 5 6 The P stipitis UC7+SUT4 strain was grown under oxygen drogenase activity Such that xylitol is accumulated. Prefer limiting conditions (50 mL of culture with a starting ODoo of ably, the yeast is recovered after the xylose in the medium is 10 in a 125 mL flask aerated at speed of 100 rpm at 30°C.). fermented to ethanol and used in Subsequent fermentations. The UC7+SUT4 strain was found to usexylose at a faster than The constructs of the invention comprise a coding the UC7 control; neither strain used all of the xylose. The 5 sequence operably connected to a promoter. Preferably, the xylitol yield of UC7+SUT4 strain (14.98 g/L) was similar to promoter is a constitutive promoter functional in yeast, or an that of the UC7 control (15.18 g/L). The UC7+SUT4 strain inducible promoter that is induced under conditions favorable had a higher ethanol specific productivity. to uptake of Sugars or to permit fermentation. Inducible pro In another example, UC7+SUT4 strain was grown in glu moters may include, for example, a promoter that is enhanced cose (7% w/w) and xylose (3% w/w) in a three liter bioreactor. 10 in response to particular Sugars, or in response to oxygen Those results indicated that the UC7+SUT4 grew at a faster rate than did the UC7 control. The UC7+SUT4 Strain had a limited conditions, such as the FAS2 promoter used in the higher biomass yield than the control. Additionally, the etha examples. Examples of other Suitable promoters include pro nol yield was higher for the UC7+SUT4 strain. The UC7+ moters associated with genes encoding P. Stipitis proteins SUT4 strain consumed both glucose and Xylose at a higher 15 which are induced in response to Xylose under oxygen limit rate and consumed all of the Sugars, whereas the control ing conditions, including, but not limited to, myo-inositol consumed only 88% of the glucose and did not appear to 2-dehydrogenase (MOR1), aminotransferase (YOD1), gua consume Xylose. Finally, the specific rate of ethanol produc nine deaminase (GAH1). These proteins correspond to pro tion on a dry cell weight basis was increased by more than tein identification numbers 64256, 35479, and 36448 on the three fold. Joint Genome Institute Pichia stipitis web site: genome.jgi In the examples, strains were developed by causing nucleic psf.org/Picst3/Picst3.home.html. acid of the invention to be introduced into a of Oxygen limiting conditions include conditions that favor the host yeast strain. This can be accomplished by any Suit fermentation. Such conditions, which are neither strictly able means. However, it is envisioned that one could obtain anaerobic nor fully aerobic, can be achieved, for example, by increased expression of the nucleic acid constructs of the 25 growing liquid cultures with reduced aeration, i.e., by reduc invention using an extrachromosomal genetic element, by ing shaking, by increasing the ratio of the culture Volume to integrating additional copies, by increasing promoter flask Volume, by inoculating a culture medium with a number strength, or by increasing the efficiency of translation through of yeast effective to provide a sufficiently concentrated initial codon optimization, all methods known to one of skill in the culture to reduce oxygenavailability, e.g., to provide an initial art. 30 cell density of 1.0 g/l dry wt of cells. It is envisioned that in addition to the particular strains that were used, any strain of S. cerevisiae or P stipitis could be By "xylose-containing material. it is meant any medium used in the practice of the invention. For example, in addition comprising Xylose, whether liquid or Solid. Suitable Xylose to P stipitis UC7, one could advantageously obtain expres containing materials include, but are not limited to, hydroly sion of the polynucleotides of the invention in P stipitis 35 sates of polysaccharide or lignocellulosic biomass Such as strains having reduced respiration capacity, such as petite corn hulls, wood, paper, agricultural by-products, and the mutants or P stipitis Shi21 (NRRLY-21971). Similarly, one like. could advantageously obtain expression of the polynucle By a “hydrolysate' as used herein, it is meant a polysac otides of the invention in a PHO-13 mutant of S. cerevisiae charide that has been depolymerized through the addition of expressing P stipitis XYL1, XYL2, and XYL3. Such a strain 40 water to form mono and oligosaccharides. Hydrolysates may was developed, as described in the examples, for use in devel be produced by enzymatic or acid hydrolysis of the polysac oping yeast strains according to the invention. Yeasts strains charide-containing material, by a combination of enzymatic may suitably be modified to include exogenous sequences and acid hydrolysis, or by an other Suitable means. expressing Xut1 and Sut4 on the same or different nucleic Preferably, the yeast strain is able to grow under conditions acid. The yeast strains may be modified to include additional 45 similar to those found in industrial sources of xylose. The advantageous features, as would be appreciated by one method of the present invention would be most economical skilled in the art. when the Xylose-containing material can be inoculated with In addition to S. cerevisiae and P Stepitis, it is envisioned the mutant yeast without excessive manipulation. By way of that other yeast species could be used to obtain yeast strains example, the pulping industry generates large amounts of according to the invention for use in the methods of the 50 cellulosic waste. Saccharification of the cellulose by acid invention. Other suitable yeast species include, without limi hydrolysis yields hexoses and pentoses that can be used in tation, Candida boidinii, Enteroramus dimorphus, Candida jeffriesii, Debaryomyces hansenii, Candida Guillermondii, fermentation reactions. However, the hydrolysate or sulfite Candida Shehatae, Brettanomyces naardensis, Candida liquor contains high concentrations of Sulfite and phenolic guillermondii, Candida lyxosophilia, Candida intermedia, 55 inhibitors naturally present in the wood which inhibit or pre Candida tenuis, Hansenula polymorpha, Kluyveromyces vent the growth of most organisms. Serially subculturing marxianus, Kluyveromyces lactis, Kluyveromyces fragilis, yeast selects for strains that are better able to grow in the Kluyveromyces thermotolerans, Pachysolen tannophilus, presence of sulfite or phenolic inhibitors. Pichia Segobiensis, Spathiaspora passalidarum and Pass 1 It is expected that yeast Strains of the present invention may isolates. 60 be further manipulated to achieve other desirable character In another aspect, the present invention provides a method istics, or even higher specific ethanol yields. For example, of fermenting Xylose in a Xylose-containing material to pro selection of mutant yeast strains by serially cultivating the duce ethanol using the yeast of the invention as a biocatalyst. mutant yeast strains of the present invention on medium con Another aspect of the present invention provides a method of taining hydrolysate may result in improved yeast with fermenting Xylose in a Xylose-containing material to produce 65 enhanced fermentation rates. Xylitol using the yeast of the invention as a biocatalyst. In this The following non limiting examples are intended to be embodiment, the yeast preferably has reduced xylitol dehy purely illustrative. US 8,105,811 B2 7 8 EXAMPLES construction with random oligonucleotides and the Reverse Transcription System Kit (Promega, Madison, Wis.). Reverse Transcription-PCR primers were designed using Primer Characterization of Putative Xylose Transporters of Pichia Express software (Applied Biosystems). RT-PCR was per stipitis formed with SYBR green PCR master mix (Applied Biosys Yeast strains, plasmids, and growth conditions. The yeast tems) and an ABI PRISM 7000 sequence detection system strains and the plasmids used in this study are listed in Table (Applied Biosystems). PCR conditions were as recom 1. S. cerevisiae EBY.VW4000 strain (MATa Ahxt 1-17 Agal2 mended by the manufacturer except that one-half of the reac Astl1 Aagt1 Amph2 Amph3 leu2-3,112 ura3-52 trp 1-289 tion Volume was used. Actin was used to normalize the rela his3-A1 MAL2-8c SUC2) (9) was kindly provided by E. tive copy numbers of each gene. All reactions were performed Boles at Heinrich-Heine-Universitat and was used for trans in triplicate. TABLE 1 List of yeast strains and plasmids

Source or Strain or plasmid Description reference Strains

S. cerevisite EBY.WW4000 MATa Ahxt1-17 Agal2 Astl1 Aagt1 Amph2 Wieczorke Amph;3 leu2-3,112 ura3-52 trp1-289 his3-A1 MAL2-8 SUC2 S. cerevisite JYB3OOS S. cerevisiae EBY.VW4000(pRS314 and This study pRS316) S. cerevisite JYB3011 S. cerevisiae EBY.VW4000(pRS314-X123 and This study pRS316) S. cerevisite JYB3110 S. cerevisiae EBY.VW4000(pRS314-X123 and This study pRS316-XUT1partial) S. cerevisite JYB3210 S. cerevisiae EBY.VW4000(pRS314-X123 and This study pRS316-XUT1) S. cerevisite JYB3310 S. cerevisiae EBY.VW4000(pRS314-X123 and This study pRS316-SUT4) Plasmids

pRS314 TRP1 CENARS Sikorski pRS316 URA3 CENARS Sikorski pRS314-X123 TRP1 CEN/ARSTDH3PSXYL1-TDH3 N TDH3PSXYL2-TDH3TDH3-PsXYL3-TDH3 pRS316-XUT1 partial URA3 CEN/ARSTDH3-PsXUT1 This study pRS316-XUT1 URA3 CENARSTDH3-PsXUT1 This study pRS316-SUT4 URA3 CEN/ARSTDH3-PsSUT4 This study formation of all the constructed plasmids. Three xylose trans Sugar-transport assay. Initial D-14CIxylose and D-C porter candidates, XUT1, XUT1 partial (corresponding to the 40 glucose (Amersham) uptake rates were measured at 30°C., sequence encoding the C-terminal region of the protein pH 5.0 as previously described (Spencer-Martins, I. a. c. U. beginning with amino acid residue 154 of SEQID NO:2), and N. (1985) Biochim. Biophys. Acta 812, 168-172). Cells were SUT4, were amplified and fused with the TDH3 promoter by harvested in the mid-exponential phase (Anm 0.8-1.0; PCR. The coding sequence of SUT4 includes a CUG codon, s0.1-0.125 g/l dry wt) by centrifugation, washed twice with which in P stipitis, specifies the serine residue of amino 45 ice-coldwater and resuspended to a cell concentration of 5-10 residue 410 of SEQ ID NO:4. This codon, which in most mg (dry weight)/ml. Kinetic parameters were determined organisms specifies a leucine, was modified for expression in using Lineweaver-Burk plots. All measurements were con S. cerevisiae using standard methods so as to specify a serine ducted at least three times, and the data reported are the residue. Each candidate had its own terminator. These frag average values. ments were then inserted into pRS316 after sequence confir 50 Transcriptional analysis of eight xylose transporter candi mation (Sikorski & Hieter (1989) Genetics 122, 19-27). dates. Eight putative xylose transporters (XUT1-7 and SUT4) Genetic manipulation and cloning of DNA were performed were identified based on genomic analysis of the genome using methods known in the art. Escherichia coli DH5OF sequence of Pichia stipitis (Jeffries et al. (2007) Nat Biotech was used as a host for plasmid preparation. 55 nol 25, 319-26) and selected for further analysis. Of these Yeast transformation was performed as previously eight putative transporters, XUT1, and SUT4 showed described (Gietz & Woods (2002) Methods Enzymol 350, increased expression in Pichia stipitis grown in Xylose-con 87-96). Yeast transformants were selected by TRP1 and taining media. These two genes were introduced into the host URA3 selectable markers and cultivated on yeast synthetic strain S. cerevisiae JYB3011 to evaluate the ability of the complete dropout (TRP-URA-) medium with carbon source 60 putative Xylose transporters to promote Xylose uptake. This (Kaiseretal. & Cold Spring Harbor Laboratory. (1994) Meth host strain was originated from S. cerevisiae EBY.VW4000 ods in yeast genetics: a Cold Spring Harbor Laboratory (Wieczorke et al. (1999) FEBS Lett 464, 123-8) and engi course manual (Cold Spring Harbor Laboratory Press, Cold neered to express PsyYL1, PsXYL2, and PsyYL3 by intro Spring Harbor, N.Y.)). Yeast cells were cultured in a 125-ml ducing pRS314-X123 (Nietal. (2007) Appl Environ Micro flask with 50 ml medium at 30° C. and 200 rpm. 65 biol 73,2061-6) (Table 1). Quantitative PCR (qPCR) Total RNA was prepared from Kinetic characteristics of Sugar uptake. XUT1 partial, each transformant and 5ug of total RNA was used for cDNA XUT1 and SUT4 were expressed in the strains S. cerevisiae US 8,105,811 B2 9 10 JYB3110, JYB 3210, and JYB3310, respectively (Table 1). PsSut2, and its Vmax for xylose uptake is about 4.4 times The expression of each transporter in its respective Strain was higher than the Vmax of Sut?. confirmed by qPCR (data not shown). These three strains Energy requirements for Sugar transport were evaluated by could grow in Xylose-containing media (FIG. 2) and in glu including the metabolic uncoupler Sodium azide. Xutl medi cose-containing media (FIG.3). Xylose and glucose transport 5 capacity of each candidate was tested using a high concen ated uptake of sugar was reduced by 96% in the presence of tration (50 mM) of labeled sugar (Table 2). The strains con Sodium azide, whereas Sut4 mediated Sugar uptake was taining Xut1 or Sut4 showed increased Xylose uptake activi reduced by about 50% (Table 4). Consequently, Xut1 is ties, with the strain expressing Xut1 having a higher uptake believed to be a high affinity Xylose-proton Symporter, and rate than that of the strain expressing Sut4 (FIG. 4). Both JYB to SUT4 appears to employ facilitated diffusion. 3210 and JYB3310 showed glucose-transporting activity, with Sut4 conferring a higher glucose uptake Velocity than TABLE 2 Xut1 (FIG. 4). Initial uptake rates of labeled Sugar (50 Kinetic analysis demonstrated that the affinity of Xut1 for mM) of each putative xylose transporter. xylose is 3-fold higher than its affinity for glucose (FIG. 4 and 15 Table 3). This is the first reported transporter having a higher Strain Substrate VE affinity for xylose. Vmax was also higher with xylose than JYB3011 (Control) Glucose 5 glucose. Although Sut4 exhibits Xylose uptake activity, its Xylose O affinity for glucose is 12 times higher than its affinity for JYB3110 (XUT1 Glucose 7 xylose (Table 3). Interestingly, Sut4 has higher Vmax with 2O partial) Xylose O Xylose than glucose. Notably, Sut4 has a higher specificity JYB3210 (XUT1) Glucose 31.8 Xylose 57.8 ratio (Vmax/Km) for both xylose and glucose than any of the JYB3310 (SUT4) Glucose S2.6 other four glucose/xylose facilitators found in Pichia stipitis Xylose 23.1 (Table 3). Table 3 compares Km values of the newly discovered 25 velocity of uptake (milimoles per minutes per milligram (dry weight) Xylose transporters to previously characterized Xylose trans TABLE 3 Kinetic parameters of yeast glucose/Xylose transporters K. Vmax Specificity mM nmol mining dw Vila K. Transporters Glucose Xylose Glucose Xylose Glucose Xylose Reference PSSut1. 1.5 + 0.1 145.0 - 1.0 45 - 1.0 132 1.0 3O.O O.9 (4) PSSlt3 O.801 103 - 3.0 3.7 O.1 87 - 2.0 4.6 O.8 (4) PSSut? 1.1 + 0.1 49.01.0 3.30.1 284.O 3.3 O.6 (4) PSSut4 1.3 O.1 16.6 O.3 1OS 4.2 122 24 80.8 7.4 This study CiGxf1 2006 48.7 6.5 s163f s25f 81.5 O.S (1) ScHxt1 10749 '880 + 8 SO.93.7 '750+ 94 O.S 0.8 (2) (3) ScHxt2 2.90.3 260 + 130 15.6 O.9 340 + 10 5.4 1.3 (2) (3) ScHxt4 6.2. O.S 170 + 120 12.O. O.9 190 + 23 1.9 1.1 (2) (3) ScHxtf 1.3 O.3 '130 + 9 11.7 O.3 '110 - 7 90.0 0.8 (2) (3) CiGXS1 O.O12 0.004 O.4 - 0.1 4.333 6.5 - 1.S 358.3 16.2 (1) PSXut1. O.91 OO1 O46 O.O2 80 - 1.0 1165.8 87.91 252.2 This study Calculated from FIG. 2 of (1) 1. Leandro, M. J.P. GonAalves, and I. Spencer-Martins, 2006. Two glucoseixylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter. The Biochemical journal 395:543-549. 2. Maier, A., B. Volker, E. Boles, and G. F. Fuhrmann. 2002. Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxt1, Hxt2, Hxt3, Hxt4, Hxté, Hxt7 or Gal2 transporters. Fems Yeast Research 2:539-550. 3. Saloheimo, A., J. Rauta, O.W. Stasyk, A.A. Sibirny, M. Penttila, and L. Ruohonen. 2007. Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases, Applied Microbiology And Biotechnology 74: 1041-1052, 4. Weierstall, T., C. P. Hollenberg, and E. Boles. 1999. Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipiis. Molecular Microbiology 31:871-883. porters. All four of the PsSut glucose/xylose facilitators show TABLE 4 similar affinities for glucose, but their affinities for xylose differ significantly. Notably, PsSut4 shows a higher specific 55 Effect of sodium azide on glucose and xylose uptake'. ity ratio (Vmax/Km) for glucose and Xylose than the speci ficity ratios of PsSut1-3. Interestingly, PsSut1 has about 78% Glucose Xylose amino acid identity with PsSut2, 3 and 4, whereas PsSut2 and Control NaNs (50 mM) Control NaNs (50 mM) PsSut4 differ from PsSut3 by one or two amino acids each JYB3110 (SUT4) 41 O.32 90 3.19 (Sut?: L279V: Sut4: L279V. A544T). Despite the high degree 60 of sequence identity, PsSut1-4 have distinct properties. JYB3310 (XUT1) 40 25 13.4 9.2 PsSut1 and PsSut3 can mediate fructose transport, whereas Velocity of uptake (nmol min mg dry weight) PsSut2 cannot, and only Pssut3 can mediate galactose trans port (Weierstall et al. (1999) Molecular Microbiol. 31:871 Effect of Expression of Sut4 in P stipitis 883). Notably, among PsSut 1-4, PsSut4 has the highest affin 65 Construction of plasmids and transformation of yeast ity and the second highest Vmax for xylose. The affinity of strains. A plasmid was constructed to contain the P stipitis PsSut4 for xylose is about three times higher than that of coding sequence for the SUT4 putative transporter under the US 8,105,811 B2 11 12 control of the P stipitis fatty acid synthase subunit alpha Evaluation of Expression of SUT4 in P stipitis in Bioreactor (Fas2) promoter Strain Descriptions. The UC7+SUT4 strain described (FAS2p SUT4 SUT4t LoxP URA3 LoxP plasmid). above (“SUT4) and the UC7+URA3 cassette strain (“con Roughly 1OO lug of the trol') described above were evaluated in a three liter bioreac FAS2p SUT4 SUT4t LoxP Ura3 LoxP plasmid was lin- 5 tor scale-up as described below. earized using Hind III and ApaI, ethanol precipitated and 3 L Bioreactor Scale-up of the SUT4 Transporter Overex resuspended in water to create afragment that would integrate pression Strain. Low oxygen bioreactor trials were used to directly into the Pichia genome. The digested construct was compare two strains in a larger scale and under more con then used in a LiAc protocol and transformed into either the trolled conditions than shake flask trials. 10 Bioreactor Cultivations. A defined minimal medium con yJML123 (Gietz & Woods (2002) Methods Enzymol 350, taining trace metal elements and vitamins was used in all 87-96)) or UC7 Pichia stipitis strains. The yJML 123 strain bioreactor cultivations. This medium was modified based on has a deletion in the XYL2 gene, which encodes the enzyme that described by Verduyn et al. (Verduyn et al. (1992)Yeast that catalyzes the conversion of Xylitol to Xylulose, allowing 8:501-517) to include urea as a nitrogen source. It had the us to create Pichia strains that will begin to accumulate Xyli 15 following composition: 2.4 g urea L'; 3 g KHPO, L', 0.5 tol. g MgSO 7HO L'; 1 ml trace element solution L'; 1 ml Transformants were selected for via growth on ScD -Ura vitamin solution L'; and 0.05 ml antifoam 289 (Sigma plates, which contain 0.62 g/L CSM-Leu-Trp-Ura (Bio A-8436) L' (Jeffries & Jin (2000) Adv Appl Microbiol 47, 101 Systems) and dextrose (2%). Colonies were then picked 221-68). For these cultivations, a starting concentration of 70 and grown overnight in ScD -Ura liquid media. Genomic 20 g L' glucose and 30 g L' xylose was used. DNA was then extracted and evaluated by PCR to confirm Cultivations were performed in 3 L New Brunswick Sci integration of the construct. As a control for these strains, a entific BioFlo 110 Bioreactors with working volumes of 2 plasmid containing only the LOXP Ura3 LOXP cassette was liters. All cultivations were performed at 30°C. Agitation was also integrated into they JML 123 and UC7 strains (yJML 123 set at 500 rpm and the pH was controlled at pH 5.0 by the and UC7 controls). 25 addition of 5 NKOH and 5 NHSO4. Aeration was controlled Fermentation of yJML 123+SUT4 Starter cultures were at a rate of 0.5 VVm which corresponded to a rate of 1 L min. established by inoculating a single colony of yJML 123+ Input gas was mixed using a gas proportioner to include 90% SUT4 strain ory.JML 123 control from recently streaked plate pure nitrogen and 10% air, for a final oxygen concentration of into 25 mL fermentation media and grown overnight. The approximately 2%. Bioreactors were inoculated to an OD of following morning, triplicate flask cultures of each transfor 30 approximately 1 and their progress followed for 74 hours. During cultivation, the SUT4 strain appeared to grow faster mant were started at an ODoo of 0.5 (s0.06 g/l dry wt of than the control. Calculated biomass rates and yields demon cells). The transformants were confirmed using PCR geno strated that SUT4 had a slightly improved growth rate (~1.2 typing. The fermentation was run under aerobic conditions fold) (0.062 vs. 0.0514 h) and biomass yield (0.151 g dry using an agitation speed of 200 rpm at 30°C. in 25 mL of cell weight/gglucose vs. 0.145g dry cell weight/gglucose, or media in a 125 mL flask in fermentation medium (Yeast 0.184 Cmmol vs. 0.177 Cmmol). As was observed previously Nitrogen Base (1.7 g/L), urea (2.4 g/L), peptone (3.6 g/L). in the shake flask experiments described above, the overall with Xylose (40 g/L) and glucose (17 g/L) as carbon Sources. difference in ethanol yield from glucose was not large. The Samples were collected for analysis roughly every 8 hours for SUT4 strain had a slightly higher (~1.11 fold) ethanol yield the first 48 hours and then once every 24 hours. 40 on glucose (0.372g ethanol/g glucose or 0.485 Cmmol) than The yJML 123+SUT4 strain was able to use both glucose the control strain (0.335 gethanol/gglucose or 0.437 Cmmol/ and xylose at a faster rate than the control strain. Neither Cmmol glucose). strain was able to use all of the xylose. They JML 123+SUT4 The major difference between these two strains is in their strain produced a higher yield of xylitol (18.45 g/L) than the kinetic rates of glucose and Xylose consumption. The SUT4 control strain (15.51 g/L). Further, the yJML 123+SUT4 45 strain consumes glucose at a much higher rate than the control strain produced a higher yield of xylitol faster than the con strain (FIG. 5). In fact, the SUT4 strain consumed all of the trol. glucose and xylose in the fermentation, while the URA3 Fermentation of UC7+SUT4. Starter cultures were estab control strain consumed approximately 88% of the glucose lished by inoculating a single colony of SUT4+UC7 strain or and did not consume enough Xylose to significantly change the UC7 control from recently streaked plates into 50 mL of 50 the Xylose concentration during the length of the fermenta fermentation media and grown overnight. The following tion. morning, triplicate flasks of each transformant were started at The specific rate of glucose consumption for the SUT4 an ODoo of 0.5 (s0.06 g/l dry wt of cells). The transformants strain (0.284 g glucose g|DCW'h' or 9.448 Cmmol were confirmed using PCR genotyping. The fermentation gDCW'h') was 3.04 fold higher than that of the URA3 was run in fermentation medium (Yeast Nitrogen Base (1.7 55 control strain (0.093 g Glucose g|DCW'h' or 3.106 Cmmol g/L), urea (2.4 g/L), peptone (3.6 g/L), and Xylose (40 g/L). gDCW'h'). The specific rates of xylose consumption could under low oxygen conditions using an agitation speed of 100 not be compared due to the fact that the URA3 control strain rpm at 30° C. in 50 mL of media in a 125 mL flask. The had not significantly begun to utilize the xylose. Further starting ODoo was 10 (s.1.25 g/l dry wt of cells). Samples bioreactor trials will be necessary to compare these rates. were collected for analysis roughly every 12 hours for the first 60 The SUT4 and control strains also exhibited different rates 60 hours and then once every 24 hours. of ethanol production. FIG. 6. shows a plot of ethanol pro Fermentation of UC7+SUT4. The UC7+SUT4 Strain was duction and glucose consumption. It should be noted that a found to use xylose at a faster rate than the UC7 control; small amount of ethanol produced by the SUT4 overexpres neither strain used all of the xylose. The xylitol yield of sion strain was due to the Xylose consumed; however the rates UC7+SUT4 Strain was similar to that of the UC7 control. The 65 and yields were calculated using the glucose only phase of UC7+SUT4 strain had a higher ethanol specific productivity fermentation. The specific rate of ethanol production for the than that of the UC7 control. SUT4 overexpression strain (0.106 gethanol g|DCW'h' or US 8,105,811 B2 13 14 4.580 Cmmol g|DCW'h') was 3.38 fold higher than that of insert. After verification, 100 ug of plasmid DNA was pre the control strain (0.031 g ETOH g|DCW'h' or 1.356 pared using a Qiagen maxiprep and digested with HpaI to Cmmol glDCW'h'). release the deletion cassette. The deletion cassette was then Creation of the Apho13 Background Strains for Transporter gel purified. Testing 5 The purified deletion cassette was then transformed into In order to test the effect of putative transporters on xylose the CEN.PK strains using a standard lithium acetate method utilization, a robust Saccharomyces cerevisiae background (Gietz & Woods (2002) Methods Enzymol350,87-96)). Out that is capable of utilizing Xylose in some capacity needed to growth was performed after the transformation for 3 hours in be built. Previous research has shown that either the overex YPD liquid media, and then transformants were selected for pression of TALI or the deletion of PHO13 promotes growth 10 on Xylose when the Pichia stipitis Xylulokinase gene is on YPD plates containing 250 mg/ml G418. After 2 days of expressed at a high level (10). The PHO13 deletion was growth, growth of putative knockouts was confirmed by re chosen for use in these strains, as more detailed kinetic data streaking on fresh G418 plates. These strains were then geno during aerobic and low oxygen cultivations is available for a typed using primers that were designed 600 bp upstream and strain created in a similar genetic background (Van Vleet etal. 15 downstream of the PHO13 open reading frame. These prim (2008) Metab. Eng. Doi10.1016/jymben. 2007.12.002). ers amplify a product in both deletion and non deletion The Strains CEN.PK. 111-27B and CEN.PK. 102-3A were strains, however in deletion strains the product is approxi chosen as the genetic backgrounds in which to create these mately 650 bp larger than in the non deletion strain. Multiple deletion strains (Entian K, Kotter P. (2007) 25 Yeast Genetic transformants for each strain were identified as containing the Strain and Plasmid Collections. In: Methods in Microbiol deletion. Transformants containing the deletion originating ogy; Yeast Gene Analysis—Second Edition, Vol. Volume 36 from CEN.PK. 111-27B were designated FPL.JHV.002 and (Ian Stansfield and Michael J R Starked), pp 629-666. Aca those originating from CEN.PK. 102-3A were designated demic Press). The CEN.PK. 111-27B carries the trp 1 and leu2 FPLUHV.OO3. mutations and the CEN.PK. 102-3A strain carries the ura3 and The xylose utilization genes (XYL1, XYL2, and XYL3) leu2 mutations. The deletion of the PHO13 in these back 25 under the control of the strong constitutive TDH3 promoter grounds will allow for the putative transporters to be tested in were then introduced to the strains via plasmid. FPL.JHV.002 the presence of the Xylose utilization genes expressed mod was transformed with the centromeric vector pRS314-X123 erately and, separately, at a high level. These transporters will (10) and designated FPL.JHV004. At the same time, FPL also be able to be tested at 2 different levels of expression. .JHV.003 was transformed with the multicopy vector pYES2 Moderate expression of the transporters will be possible 30 X123 (10) and designated FPL.JHV.005. Both of the vector using a centromeric LEU2 plasmid and high levels of expres bearing strains carry a leu2 marker to allow for testing of the sion will be possible using a multicopyLEU2 plasmid with a putative transporters. The plasmids and yeast strains are 2 um origin of replication. described in Table 6. TABLE 5

Primers Used

Primer Name Function Sequence oFPL. JHW 021 PHO13F + HpaI GATTACATGTTAACGATTGTTCGACGCAACTACCC (SEQ ID NO: 8) oFPL. JHW 022 PHO13R + HpaI GATTACATGTTAACCTTCCCCAACAAGACCGAATTG (SEO ID NO: 9)

oFPL. JHW O23 PHO13 TTAAAACAAGAATTTGGGGAG Confirmation F (SEQ ID NO: 1.O)

oFPL. JHW 024 PHO13 AAAGGTCTAATTATTCAATTTATCGAC Confirmation (SEQ ID NO: 11)

50 The PHO13 deletion cassette was amplified out of the Assessment of putative transporters by whole genome genomic DNA of S. cerevisiae CMB.JHVpho13a (Van Vleet expression array technology. Cells of Pichia stipitis were et al. (2008) Metab. Eng. Doi10.1016/jymben. 2007. 12.002) cultivated in a bioreactor with either Xylose or glucose as using the high fidelity Pfusion polymerase (NEB, carbon sources along with mineral nutrients and urea for a Finnzymes) and primers designed 400 bp upstream and 55 nitrogen source using the medium of Verduyn etal. (16). Cells downstream of the PHO13 open reading frame. This cassette were grown either under fully aerobic or oxygen limited contains the KANMX gene and has been shown previously to conditions. Cells were harvested, their mRNA was extracted confer G418 resistance to PHO13 knockout strains in S. Cer and analyzed using a whole-genome expression array evisiae (Van Vleet et al. (2008) Metab. Eng. Doi10.1016/ (NimbleGen). Transcripts corresponding to putative Sugar jymben. 2007.12.002). 60 transporters were identified and annotated. (Results not HpaI sites were added onto the ends of this amplified shown). Transcripts for seven genes (HXT2.4, HXT4, fragment for easy excision from a storage vector. The primers SUC1.5, XUT1, SUT4, XUT7 and YBR2) were induced to a used in strain creation and confirmation are listed in Table 5. higher level in cells grown on Xylose than in cells grown on The amplified fragment was ligated into the pCR4Blunt glucose. All but SUT4 were induced to their highest levels on TOPO vector and then transformed into TOP10 E. coli by a 65 Xylose under oxygen limiting conditions. Transcripts for heat shock method. Transformants were selected for onkana sevengenes (AUT1, HGT2, HUT1, AML4, RGT2, STL1 and mycin plates and digest checked to confirm the size of the SUT1) were all induced to their highest levels when cells US 8,105,811 B2 15 16 were cultivated on glucose. Transcript for one gene (SUT1) is for the purpose of description and should not be regarded as was induced to its highest level under oxygen limitation; the limiting. The use of “including.” “comprising,” or “having other six were induced to their highest levels under aerobic and variations thereofherein is meant to encompass the items conditions. The specific induction of transcripts on Xylose listed thereafter and equivalents thereofas well as additional under aerobic or oxygen limited conditions was taken as an 5 items. indicator of the effective function of this gene when cells were It also is understood that any numerical range recited cultivated on xylose. Of these genes, SUT1, SUT2 and SUT3 herein includes all values from the lower value to the upper were previously recognized and described as glucose/Xylose value. For example, if a concentration range is stated as 1% to facilitator transport proteins. 50%, it is intended that values such as 2% to 40%, 10% to It is to be understood that the invention is not limited in its 10 30%, or 1% to 3%, etc., are expressly enumerated in this application to the details of construction and the arrangement specification. These are only examples of what is specifically of components set forth in the following description. The intended, and all possible combinations of numerical values invention is capable of other embodiments and of being prac- between and including the lowest value and the highest value ticed or of being carried out in various ways. Also, it is to be enumerated are to be considered to be expressly stated in this understood that the phraseology and terminology used herein application.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 13

<21 Os SEQ ID NO 1 &211s LENGTH: 1701 &212s. TYPE: DNA <213> ORGANISM: Pichia stipitis <4 OOs SEQUENCE: 1 atgcacggtg gtggtgacgg talacgat at C acagaaatta ttgcagc.cag acgt.ct coag 60 atcgctggta agtctggtgt ggctggittta gtcgcaaact caagat ctitt Cttcatcgca 12O gtc.tttgcat ct cittggtgg attggtctac ggittacaatc aagg tatgtt cqgtcaaatt 18O

to cqg tatgt act cattctic caaagctatt ggtgttgaaa agattcaaga caatcc tact 24 O ttgcaaggtt tittgacttic tattottgaa cittggtgcct gggttggtgt Cttgatgaac 3 OO

ggitta cattg Ctgatagatt gggtcgtaag aagt cagttg ttgtcggtgt titt Ctt Cttc 360 ttcatcggtg to attgtaca agctgttgct cqtggtggta actacgact a Catcttaggit 42O

ggtagatttgtcgt.cggitat tigtgtgggt attctttct a tiggttgttgcc attgtacaat 48O gctgaagttt ct coaccaga aatticgtggit totttggttg ctittgcaa.ca attggctatt 54 O

actitt cqgta titatgatttic titactggatt accitacggta ccaact acat tdgtgg tact 6 OO ggctctggit c aaagtaaagc titcttggttg gttcc tattt gitat coaatt ggttccagct 660 ttgct Cttgg gtgttggitat cttct tcatg cctgagt ct C Caagatggitt gatgaacgaa 72O gacagagaag acgaatgttt gtc.cgttctt to Caacttgc gttcCttgag taaggaagat 78O

act cittgttcaaatggaatt cottgaaatgaaggcacaaa agttgttcga aagagaactt 84 O

totgcaaagt acttic cct ca cct coaagac ggttctgcca agagcaactt cittgattggit 9 OO

ttcaaccaat acaagtic cat gattact cac tacccaacct tcaag.cgtgt tdcagttgcc 96.O tgtttaatta tdacct tcca acaatgg act ggtgtta act tcatcttgta citatgctcca O2O

tt catct tca gttctittagg tttgtctgga aacaccattt ct cittittagc titctggtgtt O8O

gtcggitatcg to atgttcct togctaccatt coagctgttc tittgggtcga cagacittggit 14 O

agaaag.ccag titttgatttic cqgtgccatt at catgggta tttgtcactt tdttgttggct 2 OO

gcaat Cttag gt cagttcgg toggta actitt gt calaccact Coggtgctgg ttgggttgct 26 O

gttgtct tcg tttggattitt cqctatcggit tt.cggitt act Cttggggtcc atgtgcttgg 32O

gtcCttgttg cc galagt ctt Co. cattgggit ttgcgtgcta agggtgttt C tatcggtgcc 38O

tottctaact ggttgaacaa ctitcgctgtc gc catgtcta cc ccagattt tdttgctaag 4 4 O US 8,105,811 B2 17 18 - Continued gctaagttcg gtgcttacat titt cittaggit ttgatgtgta ttitt.cggtgc cqcatacgtt 15OO caattcttct gtccagaaac taagggtcgt. accttggaag aaattgatga acttitt.cggit 1560 gacaccitctg gtact tccaa gatggaaaag gaaatcCatg agcaaaagct taaggaagtt 162O ggtttgct tc aattgctcgg tgaagaaaat gctitctgaat ccgaaaacag caaggctgat 168O gtctaccacg ttgaaaaata a. 1701

<210s, SEQ ID NO 2 &211s LENGTH: 566 212. TYPE: PRT <213> ORGANISM: Pichia stipitis

<4 OOs, SEQUENCE: 2 Met His Gly Gly Gly Asp Gly Asn Asp Ile Thr Glu Ile Ile Ala Ala 1. 5 1O 15 Arg Arg Lieu. Glin Ile Ala Gly Lys Ser Gly Val Ala Gly Lieu Val Ala 25 3O

Asn Ser Arg Ser Phe Phe Ile Ala Wall Phe Ala Ser Lieu. Gly Gly Lieu. 35 4 O 45 Val Tyr Gly Tyr Asn Glin Gly Met Phe Gly Glin Ile Ser Gly Met Tyr SO 55 6 O Ser Phe Ser Lys Ala Ile Gly Val Glu Lys Ile Gln Asp Asn Pro Thr 65 70 7s 8O

Lieu. Glin Gly Lieu. Lieu. Thir Ser Ile Lieu. Glu Lieu Gly Ala Trp Val Gly 85 90 95 Val Lieu. Met ASn Gly Tyr Ile Ala Asp Arg Lieu. Gly Arg Llys Llys Ser 105 11 O

Val Val Val Gly Val Phe Phe Phe Phe Ile Gly Wall Ile Wall Glin Ala 115 12 O 125 Val Ala Arg Gly Gly Asn Tyr Asp Tyr Ile Leu Gly Gly Arg Phe Val 13 O 135 14 O

Val Gly Ile Gly Val Gly Ile Lieu. Ser Met Wall Val Pro Leu Tyr Asn 145 150 155 160

Ala Glu Wal Ser Pro Pro Glu Ile Arg Gly Ser Lieu Wall Ala Lieu. Glin 1.65 17O 17s

Gln Leu Ala Ile Thr Phe Gly Ile Met Ile Ser Tyr Trp Ile Thr Tyr 18O 185 19 O Gly Thr Asn Tyr Ile Gly Gly Thr Gly Ser Gly Glin Ser Lys Ala Ser 195 2O5

Trp Leu Val Pro Ile Cys Ile Glin Lieu Val Pro Ala Lieu. Lieu. Lieu. Gly 21 O 215 22O Val Gly Ile Phe Phe Met Pro Glu Ser Pro Arg Trp Lieu Met Asn. Glu 225 23 O 235 24 O

Asp Arg Glu Asp Glu. Cys Lieu. Ser Wall Lieu. Ser Asn Lieu. Arg Ser Lieu. 245 250 255

Ser Lys Glu Asp Thir Lieu Val Glin Met Glu Phe Lieu. Glu Met Lys Ala 26 O 265 27 O Glin Llys Lieu. Phe Glu Arg Glu Lieu. Ser Ala Lys Tyr Phe Pro His Leu 285

Glin Asp Gly Ser Ala Lys Ser Asn Phe Lieu. Ile Gly Phe Asin Glin Tyr 29 O 295 3 OO Lys Ser Met Ile Thr His Tyr Pro Thr Phe Lys Arg Val Ala Val Ala 3. OS 310 315 32O

Cys Lieu. Ile Met Thr Phe Glin Glin Trp Thr Gly Wall Asn. Phe Ile Lieu 3.25 330 335 US 8,105,811 B2 19 20 - Continued

Tyr Tyr Ala Pro Phe Ile Phe Ser Ser Luell Gly Lell Ser Gly Asn. Thir 34 O 345 35. O

Ile Ser Lieu. Lieu Ala Ser Gly Val Wall Gly Ile Wall Met Phe Lieu Ala 355 360 365

Thir Ile Pro Ala Val Lieu. Trp Val Asp Arg Luell Gly Arg Lys Pro Wall 37 O 375

Lieu. Ile Ser Gly Ala Ile Ile Met Gly Ile Cys His Phe Wall Wall Ala 385 390 395 4 OO

Ala Ile Lieu. Gly Glin Phe Gly Gly Asn Phe Wall Asn His Ser Gly Ala 4 OS 415

Gly Trp Val Ala Val Val Phe Val Trp Ile Phe Ala Ile Gly Phe Gly 425 43 O

Tyr Ser Trp Gly Pro Cys Ala Trp Wall Luell Wall Ala Glu Wall Phe Pro 435 44 O 445

Lieu. Gly Lieu. Arg Ala Lys Gly Val Ser Ile Gly Ala Ser Ser Asn Trp 450 45.5 460

Lieu. Asn. Asn. Phe Ala Wall Ala Met Ser Thir Pro Asp Phe Wall Ala Lys 465 470 48O

Ala Lys Phe Gly Ala Tyr Ile Phe Luell Gly Luell Met Ile Phe Gly 485 490 495

Ala Ala Tyr Val Glin Phe Phe Cys Pro Glu Thir Gly Arg Thir Lieu. SOO 505

Glu Glu Ile Asp Glu Lieu. Phe Gly Asp Thir Ser Gly Thir Ser Lys Met 515 525

Glu Lys Glu Ile His Glu Gln Lys Luell Glu Wall Gly Luell Lieu. Glin 53 O 535 54 O

Lieu. Lieu. Gly Glu Glu Asn Ala Ser Glu Ser Glu Asn Ser Ala Asp 5.45 550 555 560 Val Tyr His Val Glu Lys 565

<210s, SEQ ID NO 3 &211s LENGTH: 1653 &212s. TYPE: DNA <213> ORGANISM: Pichia stipitis

<4 OOs, SEQUENCE: 3 atgtcc toac aagatttacc Ctcgggtgct Caaac Co. Cala tcgatggttc t to catcc to 6 O gaagataaag ttgagcaaag titcgt.cctica aatagccaaa gtgatttagc t to tatt coa 12 O gcaa.caggta t caaagccta t ct cittggitt tgtttcttct gcatgttggit tgcct tcggit 18O ggct tcgt at t cqgtttcga taccgg tact attitcc.ggitt to Cittaatat gtctgatttic 24 O ctitt coagat ttggtcaaga tggttctgaa ggaaaat att tgtctgatat cagagtcggit 3OO ttgattgttt coatttittaa cattggttgt gcaattggtg gtattitt cot ttctaagata 360 ggagatgttt acggtagaag aattggitatic attt cagcta tggttgtcta tgtcgt.cggit ataatcatcc agatct cqtc ccalagataag tggitat caac ttacaattgg acgtggagtt acaggattag Ctgttggtac ttgtc.t.c caa tgttcattag tgaaagtgct 54 O Cccalagcatt tagaggtac tittggtatac tgttaccaat tatgcatcac cittagg tatt tt cattggitt actgtgtcac titatggaacc aaagatttaa atgattcaag acaatggaga 660 gttc ctittgg gtt tatgttt CCtttgggct attitt cittag ttgttggitat gttggctatg 72 O ccagaatc cc caagatt citt gattgaaaag aagagaatcg aagaa.gc.cala gaagt cc citt US 8,105, 811 B2 21 - Contin lued gcaagatc.ca acaagttgtc tccagaagat cCaggtgtct acactgaagt t caattgatt 84 O

Caagctggta ttgacagaga agctgctgca ggttctgctt cgtggatgga attgattacc 9 OO ggtaalaccag ctattitt cag aagagittatc atgggaatta t cittacagtic tittgcagcaa 96.O ttaactggtg toalactattt cit to tact act ggalactacaa tct tccaagc tgttggtttg caagattic ct tccagactitc CatCatctta ggtacagtica act t t citt to tacatttgtt gg tatttggg c cattgaaag atttggaaga agacaatgtt tgttagt cqg ttctgctggit 14 O atgttcgttt gttt catcat ttact ctdtc attgg tacaa ct catttgtt cattgatgga 2OO gtag tagata acgacaacac cc.gtcaactg tctggtaatg ctatoat citt tat cacttgt 26 O ttgttcatct tottctittgc Ctgtacttgg gctggaggtg tttitt acaat cattt cogala 32O toatat CCat tgagaatcag atccaaggct atgtctattg c cactg.ccgc taactggatg tggggtttct tgattt catt ctgcacticca tt cattgtta acgc.cat caa cittcaagttc 44 O ggctttgttgt ttactggttg tttgctottt togttcttct atgtc tactt Ctttgtcagc SOO gaalaccalaag gtttgtcgtt ggaagaagtt gatgagttgt acgctgaggg aattgcacca 560 tggaaatc.cg gtgcatgggt tcotcottct gcacaacaac aaatgcaaaa c to tact tatt

caaaagagca agagcaagtt tag 653

<210s, SEQ ID NO 4 &211s LENGTH: 550 212. TYPE : PRT &213s ORGANISM: Pichia stipitis

<4 OOs, SEQUENCE: 4 Met Ser Ser Glin Asp Leu Pro Ser Gly Ala Glin Thr Pro Ile Asp Gly 1. 1O 15

Ser Ser Ile Lieu. Glu Asp Llys Val Glu Glin Ser Ser Ser Ser Asn. Ser 25 3O

Glin Ser Asp Lieu Ala Ser Ile Pro Ala Thr Gly Ile Lys Ala Tyr Lieu. 35 4 O 45

Lell Wall Phe Phe Cys Met Leu Wall Ala Phe Gly Gly Phe Wall Phe SO 55 6 O

Gly Phe Asp Thr Gly Thr Ile Ser Gly Phe Leu Asn Met Ser Asp Phe 65 70 7s

Lell Ser Arg Phe Gly Glin Asp Gly Ser Glu Gly Llys Tyr Lieu. Ser Asp 85 90 95

Ile Arg Wall Gly Lieu. Ile Val Ser Ile Phe Asn Ile Gly Cys Ala Ile 105 11 O

Gly Gly Ile Phe Lieu. Ser Lys Ile Gly Asp Wall Arg Ile 115 12 O 125

Gly Ile Ile Ser Ala Met Wal Wall Tyr Val Val Gly Ile Ile Ile Glin 13 O 135 14 O

Ile Ser Ser Glin Asp Llys Trp Tyr Gln Lieu. Thir Ile Gly Arg Gly Val 145 150 155 160

Thir Gly Luell Ala Val Gly Thr Val Ser Wall Lieu Ser Pro Met Phe Ile 1.65 17O 17s

Ser Glu Ser Ala Pro Llys His Lieu. Arg Gly Thr Lieu Val Tyr 18O 185 19 O

Glin Luell Cys Ile Thr Lieu. Gly Ile Phe Ile Gly Tyr Cys Val Thr Tyr 195 2O5

Gly Thir Asp Lieu. Asn Asp Ser Arg Glin Trp Arg Val Pro Lieu. Gly 21 O 215 22O US 8,105,811 B2 23 24 - Continued Lell Phe Lieu. Trp Ala Ile Phe Lieu Wal Wall Gly Met Leu Ala Met 225 23 O 235 24 O

Pro Glu Ser Pro Arg Phe Lieu. Ile Glu Lys Llys Arg Ile Glu Glu Ala 245 250 255

Ser Lieu Ala Arg Ser Asn Llys Lieu. Ser Pro Glu Asp Pro Gly 26 O 265 27 O

Wall Thir Glu Wall Glin Lieu. Ile Glin Ala Gly Ile Asp Arg Glu Ala 27s 285

Ala Ala Gly Ser Ala Ser Trp Met Glu Lieu. Ile Thir Gly Pro Ala 29 O 295 3 OO

Ile Phe Arg Arg Val Ile Met Gly Ile Ile Lieu. Glin Ser Luell Glin Glin 3. OS 310 315

Lell Thir Gly Val Asn Tyr Phe Phe Thir Thir Ile Phe Glin 3.25 330 335

Ala Wall Gly Lieu. Glin Asp Ser Phe Glin. Thir Ser Ile Ile Luell Gly Thr 34 O 345 35. O

Wall Asn Phe Lieu. Ser Thir Phe Wall Gly Ile Trp Ala Ile Glu Arg Phe 355 360 365

Gly Arg Arg Glin Cys Lieu. Lieu Val Gly Ser Ala Gly Met Phe Val Cys 37 O 375

Phe Ile Ile Tyr Ser Val Ile Gly Thir Thir His Lell Phe Ile Asp Gly 385 390 395 4 OO

Wall Wall Asp Asn Asp Asn. Thir Arg Glin Ser Ser Gly Asn Ala Met Ile 4 OS 41O 415

Phe Ile Thir Cys Lieu Phe Ile Phe Phe Phe Ala Thir Trp Ala Gly 425 43 O

Gly Wall Phe Thir Ile Ile Ser Glu Ser Tyr Pro Lell Arg Ile Arg Ser 435 44 O 445

Ala Met Ser Ile Ala Thir Ala Ala Asn Trp Met Trp Gly Phe Lieu. 450 45.5 460

Ile Ser Phe Cys Thr Pro Phe Ile Wall Asn Ala Ile Asn Phe Llys Phe 465 470 47s 48O

Gly Phe Wall Phe Thr Gly Cys Lieu Lieu. Phe Ser Phe Phe Val Tyr 485 490 495

Phe Phe Wall Ser Glu Thir Lys Gly Luell Ser Luell Glu Glu Wall Asp Glu SOO 505 51O

Lell Ala Glu Gly Ile Ala Pro Trp Llys Ser Gly Ala Trp Wall Pro 515 525

Pro Ser Ala Glin Glin Gln Met Glin Asn. Ser Thr Tyr Gly Ala Glu Thir 53 O 535 54 O

Lys Glu Glin Glu Glin Wall 5.45 550

<210s, SEQ ID NO 5 &211s LENGTH: 1213 &212s. TYPE: DNA <213> ORGANISM: Pichia stipitis <4 OOs, SEQUENCE: 5 gttatgggaa accalattggg taccaaaagt gcaaaattta tatatggata caaaaagtta 6 O tattagaatt actgtttaga tgc gaataga tgttgat cat ttgtaatagt cctag tatga 12 O taccaaaagt gaccgtgggt citat catgat agggtggaga tgat ctittga tatt ccaaag 18O caaaagtgtt CCCttaalacc agtttagact gaaacaa.ccg aatgtaatca ggg tagatga 24 O gaaggcatta agctgttggtg tittggct caa aaagagattic tacacalatat tggactittga 3OO

US 8,105,811 B2 31 32 - Continued ttgattgttt coatttittaa cattggttgt gcaattggtg g tattitt cot ttctaagata 360 ggagatgttt acggtagaag aattggitatic atttcagcta tigttgtata C9tcgt.cggit 42O attatcatcc agatct cqtc ccaagacaag togg taccaac ttacaattgg acgtggagtt 48O acaggattag ctgttgg tac tdttt cagtg ttgtc. tccaa tott cattag togaaagtgct 54 O c caaag catt toga gagg tac tittggtatac tdttaccaat tatgtat cac cittagg tatt 6OO tt cattggitt actgtgtcac titatggaacc aaagatttaa atgattcaag acaatggaga 660 gttc ctittgg gct tatgctt cctittgggct attitt cittag ttgtcggitat gttggctatg 72 O ccagaatc cc caagattctt aattgaaaag aagagaatcg aagaa.gc.caa gaagt cc citt 78O gcaagatcca acaagttatc. tcc.ggaagat coaggtgtct acactgaact t caattgatt 84 O

Caggctggta ttgacagaga agctgctgca ggttctgctt citggatgga attgat cact 9 OO ggtaagc.cag c tattitt cag aagagittatc atgggaatta t cittgcagtic tittgcaacaa 96.O ttaactggtg tdaact attt Cttct attac ggaactacaa tottccaagc tigttggtttg O2O caagattic ct tccagactitc catcatctta ggtacagtica actitt ctitt c tacatttgtt O8O gg tatttggg C cattgaaag atttggaaga agacaatgtt tittagt cqg ttctgctggit 14 O atgttcgttt gttt catcat titact cogitt attgg tacaa ct catttgtt cattgatgga 2OO gtag tagata acgacaacac ccgtcaactg. tctggtaatig citatgat citt tat cacttgt 26 O ttgttcatct tcttctittgc ctd tacatgg gctggaggtg tttittaccat cattt cogala 32O t catat coat tdagaatcag atccaaggca atgtctattg c tactgctgc taactggatg 38O tgggg.cttct tdattt cott ctdcactic catt cattgtta atgc.cat caa cittcaagttc 44 O ggctttgttgt ttactggttgttt actictitt togttctitct atgtc tactt citttgtcagc SOO gaalaccalaag gtttgtcgtt ggaagaagtt gatgagttgt acgctgaagg tattgcacca 560 tggaagttctg gtgcatgggit to citccttct gcc caacaac aaatgcaaaa citccactitat 62O ggtgc.cgaag caaaagagca agagcaagtt tagstt 656

We claim: 11. The strain of claim 8, wherein the strain exhibits 1. A nucleic acid construct comprising a coding sequence increased Xylitol production relative to a control yeast lacking operably linked to a promoter not natively associated with the the construct. coding sequence, the coding sequence encoding a glucose/ 45 12. The strain of claim 11 wherein the strain has reduced Xylose transporter polypeptide having at least 95% amino Xylitol dehydrogenase activity Such that xylitol accumulates. 13. The strain of claim 8, wherein the yeast strain is a acid identity to SEQID NO:4, wherein the polypeptide com Pichia stipitis. prises a serine residue at the amino acid position correspond 14. The strain of claim 8, wherein the yeast strain is a ing to position 544 of SEQID NO:4. Saccharomyces cerevisiae. 2. The construct of claim 1, wherein the promoter is a 50 15. A method of producing ethanol from the fermentation constitutive promoter. of xylose comprising: culturing the yeast strain of claim 8 in 3. The construct of claim 1, wherein the promoter is induc Xylose-containing material under Suitable conditions for a ible under oxygen limiting conditions. period of time sufficient to allow fermentation of at least a 4. The construct of claim 1, wherein the polypeptide com portion of the xylose to ethanol. prises SEQID NO:4. 55 16. The method of claim 15, wherein the xylose-containing 5. The construct of claim 1, wherein the coding sequence material further comprises glucose. comprises at least one of the sequences shown in FIG. 1. 17. A method of producingxylitol from the fermentation of 6. The construct of claim 1, wherein the coding sequence Xylose comprising the step of culturing the yeast strain of comprises SEQID NO:3. claim 9 in Xylose-containing material under Suitable condi 7. A vector comprising the construct of claim 1. 60 tions for a period of time sufficient to allow fermentation of at 8. A yeast strain comprising the construct of claim 1. least a portion of the xylose to xylitol. 9. The strain of claim 8, wherein the strain exhibits 18. The construct of claim 2, wherein the constitutive pro increased uptake of xylose relative to a control yeast lacking moter is TDH3. the construct. 19. The construct of claim 3, wherein the promoter is 10. The strain of claim 8, wherein the strain has a higher 65 selected from the group consisting of FAS2, MOR1, YOD1 specific rate of ethanol production relative to a control yeast and GAH1. lacking the construct.