CLONING AND CHARACTERIZATION OF L-ARABINOSE TRANSPORTERS FROM NON-CONVENTIONAL YEAST

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S. C. 119(e) to U.S. Provisional

Patent Application No. 60/810,274 entitled "CLONING AND CHARACTERIZATION OF L-ARABINOSE TRANSPORTERS FROM NON-CONVENTIONAL YEAST," filed June 2, 2006, the disclosure of which is hereby incorporated by reference in its entirety.

CONTRACTUAL ORIGIN

[0002] The United States Government has rights in this invention under Contract No.

DE-AC36-99GO10337 between the United States Department of Energy and the National Renewable Energy Laboratory, a Division of the Midwest Research Institute.

BACKGROUND

[0003] Fuel ethanol is a suitable alternative to fossil fuels. Ethanol may be produced from plant biomass, which is an economical and renewable resource that is available in large amounts. Examples of biomass include agricultural feedstocks, paper wastes, wood chips and so on. The sources of biomass vary from region to region based on the abundance of natural or agricultural biomass that is available in a particular region. For example, while sugar cane is the primary source of biomass used to produce ethanol in Brazil, corn-derived biomass, such as corn starch and corn fiber, is a large source of biomass used to produce ethanol in the United States. Other agricultural feedstocks include, by way of example: straw; grasses such as switchgrass; grains; and any other cellulose or starch-bearing material. [0004] A typical biomass substrate contains from 35-45% cellulose, 25-40% hemicellulose, and 15-30% lignin, although sources may be found that deviate from these general ranges. As is known in the art, cellulose is polymer of glucose subunits, and hemicellulose contains mostly xylose. Arabinose is also a significant fermentable substrate that may be found in biomass, such as corn fiber and many herbaceous crops. Other researchers have investigated the utilization of arabinose and hemicellulose, as reported by Hespell, R. B. 1998. Extraction and characterization of hemicellulose from the corn fiber

produced by corn wet-milling processes. J. Agric. Food Chem. 46:2615-2619, and McMillan, J. D., and B. L. Boynton. 1994. Arabinose utilization by xylose-fermenting yeasts and fungi. Appl. Biochem. Biotechnol. 45-46:569-584. The two most abundant types of pentose that exist naturally are D-xylose and L-arabinose.

[0005] It is problematic that most of the naturally available ethanol-producing microorganisms are only capable of utilizing hexose sugar, such as glucose. This is confirmed by a review of the art, such as is reported by Barnett, J. A. 1976. The utilization of sugars by yeasts. Adv. Carbohydr. Chem. Biochem. 32: 125-234. Many types of yeast, especially Saccharomyces cerevisiae and related species, are very effective in fermenting glucose-based feedstocks into ethanol through anaerobic fermentation. However, these glucose-fermenting yeasts are unable to ferment xylose or L-arabinose, and are unable to grow solely on these pentose sugars. Although some other yeast species, such as Pichia stipitis and Candida shehatae, may ferment xylose to ethanol, they are not as effective as Saccharomyces for fermentation of glucose and have a relatively low level of ethanol tolerance. Thus, the present range of available yeast are not entirely suitable for large scale industrial production of ethanol from biomass.

[0006] Most bacteria, including E. coli and Bacillus subtilis, utilize L-arabinose for aerobic growth, but they do not ferment L-arabinose to ethanol. Other microorganisms, such as Zymononas mobilis, have also been genetically modified to produce ethanol from hexose or pentose. This has been reported, for example, in Deanda, K., M. Zhang, C. Eddy, and S. Picataggio. 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering. Appl. Environ. Microbiol. 62:4465-4470; and Zhang, M., C. Eddy, K. Deanda, M. Finkelstein, and S. Picataggio. 1995 Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science 267:240-243. However, it remains the case that low alcohol tolerance of these non-yeast microorganisms limits their utility in the ethanol industry.

[0007] Much effort has been made over the last decade or so, without truly overcoming the problem of developing new yeast strains that ferment xylose to generate ethanol. Such efforts are reported, for example, in Kδtter, P., R. Amore, C. P. Hollenberg, and M. Ciriacy. 1990. Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant. Curr. Genet. 18:493-500; and Wahlbom, C. F., and B. Hahn-Hagerdal. 2002. Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae, Biotechnol.

Bioeng. 78: 172-178. Recent studies have been conducted on yeast strains that may ferment arabinose. Sedlak, M., and N. W. Ho. 2001. Expression ofii. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae, Enzyme Microb. Technol. 28: 16-24 discloses the expression of an ii. coli araBAD operon encoding enzymes for metabolizing L-arabinose in Saccharomyces cerevisiae. Although this strain expresses araA, araB and araD proteins, it is incapable of producing ethanol. [0008] United States Patent Application 10/983,951 by Boles and Becker discloses the creation of a yeast strain that may ferment L-arabinose. However, the overall yield is relatively low, at about 60% of theoretical value. The rate of arabinose transport into S. cerevisiae may be a limiting factor for complete utilization of the pentose substrate. Boles and Becker attempted to enhance arabinose uptake by over expressing the GAL2-encoded galactose permease in S. cerevisiae. However, the rate of arabinose transport using galactose permease was still much lower when compared to that exhibited by non-conventional yeast such as Kluyveromyces marxianus. Another limitation that may have contributed to the low yield of ethanol in the modified strain of Becker and Boles is the poor activity of the L- arabinose isomerase encoded by the bacterial araA gene. Although Becker and Boles used an araA gene from B. subtilis instead of one from E. coli, the specific activity of the enzyme was still low. Other workers in the field have reported that low isomerase activity is a bottleneck in L-arabinose utilization by yeast.

[0009] There remains a need for new arabinose-fermenting strains that are capable of producing ethanol at high yield. There is further a need to identify novel arabinose transporter for introduction into Saccharomyces cerevisiae to boost the production of ethanol from arabinose.

[0010] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.

SUMMARY

[0011] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-

described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

[0012] Systems, tools and methods are provided for the identification of yeast strains having efficient, single component L-arabinose transport mechanisms. These strains were identified by sequentially screening yeast strains for L-arabinose utilization, ameability of L- arabinose utilizing yeast strains to genetic and biochemical manipulation, autotrophy, and specific L-arabinose transport rates. Yeast strains identified using the systems, tools and methods of the embodiment were then further characterized to identify novel arabinose transporter genes.

[0013] As such, the presently disclosed instrumentalities provide cloned and characterized novel arabinose transporter genes, termed KmLATland PgLAT2, from two non- conventional yeast species, Kluyveromyces marxianus and Pichia guilliermondii (also known as Pichia guilliermondii), respectively. It is disclosed herein that both Kluyveromyces marxianus and Pichia guilliermondii are efficient utilizers of L-arabinose, which renders them ideal sources for cloning L-arabinose transporter genes.

[0014] The KmLATl gene may be isolated using functional complementation of an adapted S. cerevisiae strain that could not grow on L-arabinose because it lacked sufficient L- arabinose transport activity. KmLatl protein has a predicted length of 556 amino acids encoded by a single ORF of 1668 bp. It is a transmembrane protein having high homology to sugar transporters of many different yeast species. When KmLatl is expressed in S. cerevisiae, transport assays using labeled L-arabinose show that this transporter has the kinetic characteristics of a low affinity arabinose transporter, with K _m = 230 mM and V _max = 55 nmol/mg-min. Transport of L-arabinose by KmLatl is not significantly inhibited by common uncoupling agents but is out-competed by glucose, galactose, xylose, and maltose. [0015] The PgLAT2 gene may be isolated using the technique of differential display from Pichia guilliermondii. The PgLAT2 gene has an ORF of 1617 nucleotides encoding a protein with a predicted length of 539 amino acids. It is also predicted to be a transmembrane protein and shows high homology to sugar transporters of many different yeast species. When PgLat2 is expressed in S. cerevisiae, transport assays show that this transporter has almost identical L-arabinose transport kinetics as that of wildtype Pichia guilliermondii. The PgLat2 transporter when expressed in S. cerevisiae has a K _m of 0.07 mM and V _m a _x of 18 nmol/mg-min for L-arabinose transport. Inhibition experiments show significant inhibition of the PgLat2 transporter by protonophores (e.g., NaN3, DNP, and CCP) and H+-adenosine triphosphatase (ATPase) inhibitors (e.g., DESB and DCCD) similar to inhibition in wildtype

P. guilliermondii. Competition experiments show that L-arabinose uptake by the PgLat2 transporter is inhibited by glucose, galactose, xylose and to a lesser extent by maltose. [0016] The transport kinetics of S. cerevisiae Gal2p have been measured and compared to those of KmLatl. The S. cerevisiae GAL2 gene (SEQ ID NO 5) under control of a TDH3 promoter exhibits 28 times greater (8.9 nmol/mg-min) L-arabinose transport rate as compared to GAL2 gene under control of a ADHl promoter. The GAL2-encoded permease (SEQ ID NO 6) shows a K _m of 550 mMand a V _max of 425 nmol/mg-min for L-arabinose transport and a K _m of 25 vaM and a V _max of 76 nmol/mg-min for galactose transport. Although L-arabinose transport by both KmLATl and GAL2 encoded permeases is out- competed by glucose or galactose, the inhibitory effects of glucose or galactose are greater on the GAL2 encoded permease than on the KmLATl encoded transporter. [0017] It is further disclosed here that a S. cerevisiae strain may be transformed with different combinations of the KmLATl and PgLAT2 transporter genes and a plasmid carrying the GAL2 gene native to S. cerevisiae. The doubling time for the PgLat2p and Gal2p co- expressing cells grown on L-arabinose is markedly shorter than that of the cells expressing only Gal2p, suggesting that L-arabinose uptake may have been enhanced in these cells. In addition, the PgLat2p and Gal2p co-expressing cells appear to grow to a higher optical density at saturation, suggesting that this strain may be able to utilize the L-arabinose in the medium more completely. This conclusion is supported by HPLC analysis which shows significantly less residual L-arabinose in the culture of cells expressing PgLat2p and Gal2p. [0018] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Exemplary embodiments are illustrated in referenced figures of the drawings.

It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

[0020] Figure 1 shows phenotypes of L-arabinose-negative mutants obtained from A. adeninivorans . The numbers of mutants in each class are indicated in the boxes. The grey shaded box indicates the mutant class in which transport is expected to have been impacted. [0021] Figure 2 shows phenotypes of L-arabinose-negative mutants obtained from D. hansenii The numbers of mutants in each class are indicated in the boxes. The

grey shaded box indicates the mutant class in which transport is expected to have been impacted.

[0022] Figure 3 shows phenotypes of L-arabinose-negative mutants obtained from P. guilliermondii. The numbers of mutants in each class are indicated in the boxes. The grey shaded box indicates the mutant class in which transport is expected to have been impacted.

[0023] Figure 4 shows testing for impurities in L-(l- ¹⁴ C)arabinose, 0.1 μl of D-(I-

¹⁴ C)galactose (lane 1), D-(l- ¹⁴ C)xylose (lane 2) and L-(l- ¹⁴ C)arabinose (lane 3) were separated on Whatman K6 TLC plates. The positions of migration of other related compounds on this TLC system are indicated.

[0024] Figure 5 shows the identification of L-(l- ¹⁴ C)arabinose. Sample incubated without galactose dehydrogenase (lane 1); Sample incubated with galactose dehydrogenase

(lane 2). The TLC was performed as described in Materials and Methods of Example 1.

[0025] Figure 6 shows the Eadie-Hofstee plots of L-arabinose transport. Initial rates of labeled L-arabinose uptake (0.065-592.2 mM) by L-arabinose grown cells were determined. A: A. adeninivorans . B: D. hansenii Yar.fabryii. C: K. marxianus. D: P. guilliermondii.

[0026] Figure 7 shows the fungal pathway for L-arabinose metabolism.

[0027] Figure 8 shows the relationship between KmLATl and other transporters based on the neighbor-joining method (Saitou and Nei 1987).

[0028] Figure 9 shows the DNA (SEQ ID NO. 1) sequence of Kluyveromyces marxianus KmLATl, and the predicted protein sequence (SEQ ID NO. 2).

[0029] Figure 10 shows the library insert from genomic K. marxianus DNA complements adapted S. cerevisiae for growth on L-arabinose. Cloning into the library expression vector is at the indicated BamHI restriction sites. The black block arrow is the L- arabinose transporter ORF responsible for complementation (KmLATl). The block arrow with vertical stripes is the interrupted transporter ORF. The block arrow with the horizontal stripes is an un-related ORF ligated in place gratuitously during library construction. The

Sau3AI restriction site where the transporter ORF was interrupted is shown. The primer used for PCR based genomic walking in K marxianus is shown.

[0030] Figure 11 shows the growth curve of S. cerevisiae expressing KmLATl (δ),

GAL2 (■) or a control vector (♦) on 2% L-arabinose.

[0031] Figure 12 (A): Eadie-Hofstee plot of L-arabinose uptake by KmLatl (♦) or

Gal2 (■) expressed in S. cerevisiae grown on 2% L-arabinose. (B): Comparison of Eadie-

Hofstee plots of KmLatl expressed in S. cerevisiae (♦) and wild type transport activity of K. marxianus (δ) both grown on 2% L-arabinose.

[0032] Figure 13 shows the DNA (SEQ ID NO. 3) sequence of Pichia guilliermondii

PgLAT2, and the predicted protein sequence (SEQ ID NO. 4).

[0033] Figure 14 shows the induction of L-arabinose transport in P. guilliermondii.

Uptake of 13 niM labeled sugar was assayed for cells grown in minimal media containing 2%

L-arabinose, D-galactose or D-xylose. White bars indicate labeled L-arabinose transport.

Black bars indicate labeled galactose transport. Bars with vertical stripes indicate labeled xylose transport.

[0034] Figure 15 shows the sugar transport competition analysis in P. guilliermondii grown in minimal L-arabinose medium.

[0035] Figure 16 shows the transport kinetics of L-arabinose by the PgLat2 transporter expressed in S. cerevisiae. Open triangles indicate transport for wild type P. guilliermondii grown on L-arabinose. Black diamonds indicate transport for PgLat2 expressed in S. cerevisiae grown on L-arabinose.

[0036] Figure 17 shows comparison of the growth curves in 0.2% L-arabinose for S. cerevisiae cells expressing either Gal2p alone or both Gal2p and PgLat2. The maximum growth density and growth rate are significantly enhanced in the strain expressing both Gal2p and PgLat2.

DETAILED DESCRIPTION

[0037] There will now be shown and described systems, methods and tools for identifying single component, high efficiency arabinose transport proficient yeast strains. These identified yeast stains provide the source material for identifying and characterizing single component arabinose transporter genes identified herein. In particular, several highly efficient arabinose transport genes, for example KmLATl and PgLAT2, are provided. The identified transporter genes, for example KmLATl and PgLAT2, may be inserted into a host for metabolizing arabinose and, under conditions described herein, produce ethanol in high yield.

[0038] In the discussion below, parenthetical mention is made to publications from the references section for a discussion of related procedures that may be found useful from a perspective of one skilled in the art. This is done to demonstrate what is disclosed by way of non-limiting example.

[0039] The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure:

[0040] "Amino acid" refers to any of the twenty naturally occurring amino acids as well as any modified amino acid sequences. Modifications may include natural processes such as posttranslational processing, or may include chemical modifications which are known in the art. Modifications include but are not limited to: phosphorylation, ubiquitination, acetylation, amidation, glycosylation, covalent attachment of flavin, ADP-ribosylation, cross linking, iodination, methylation, and the like.

[0041] "Antibody" refers to a generally Y-shaped molecule having a pair of antigen binding sites, a hinge region and a constant region. Fragments of antibodies, for example an antigen binding fragment (Fab), chimeric antibodies, antibodies having a human constant region coupled to a murine antigen binding region, and fragments thereof, as well as other well known recombinant antibodies are included in this definition.

[0042] "Antisense" refers to polynucleotide sequences that are complementary to target "sense" polynucleotide sequence.

[0043] "Biomass" refers collectively to organic non-fossil material. "Biomass" in the present disclosure refers particularly to plant material that is used to generate fuel, such as ethanol. Examples of biomass includes but are not limited to corn fiber, dried distiller's grain, jatropha, manure, meat and bone meal, miscanthus, peat, plate waste, landscaping waste, maize, rice hulls, silage, stover, maiden grass, switchgrass, whey, and bagasse from sugarcane.

[0044] "Complementary" or "complementarity" refers to the ability of a polynucleotide in a polynucleotide molecule to form a base pair with another polynucleotide in a second polynucleotide molecule. For example, the sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'. Complementarity may be partial, in which only some of the polynucleotides match according to base pairing, or complete, where all the polynucleotides match according to base pairing.

[0045] "Expression" refers to transcription and translation occurring within a host cell. The level of expression of a DNA molecule in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of DNA molecule encoded protein produced by the host cell (Sambrook et al., 1989,

Molecular cloning: A Laboratory Manual, 18.1- 18.88).

[0046] "Fusion protein" refers to a first protein attached to a second, heterologous protein. Preferably, the heterologous protein is fused via recombinant DNA techniques, such

that the first and second proteins are expressed in frame. The heterologous protein may confer a desired characteristic to the fusion protein, for example, a detection signal, enhanced stability or stabilization of the protein, facilitated oligomerization of the protein, or facilitated purification of the fusion protein. Examples of heterologous proteins useful as fusion proteins include molecules having full-length or partial protein sequence of KmLatl or PgLat2. Further examples include peptide tags such as histidine tag (6-His), leucine zipper, substrate targeting moieties, signal peptides, and the like. Fusion proteins are also meant to encompass variants and derivatives of KmLatl or PgLat2 polypeptides that are generated by conventional site-directed mutagenesis and more modern techniques such as directed evolution, discussed infra.

[0047] "Genetically engineered" refers to any recombinant DNA or RNA method used to create a prokaryotic or eukaryotic host cell that expresses a protein at elevated levels, at lowered levels, or in a mutated form. In other words, the host cell has been transfected, transformed, or transduced with a recombinant polynucleotide molecule, and thereby been altered so as to cause the cell to alter expression of the desired protein. Methods and vectors for genetically engineering host cells are well known in the art; for example various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates). Genetic engineering techniques include but are not limited to expression vectors, targeted homologous recombination and gene activation (see, for example, U.S. Patent No. 5,272,071 to Chappel) and trans activation by engineered transcription factors (see, for example, Segal et al., 1999, Proc Natl Acad Sci USA 96(6):2758-63).

[0048] "Homology" refers to a degree of similarity between polynucleotides, having significant effect on the efficiency and strength of hybridization between polynucleotide molecules. The term also refers to a degree of similarity between polypeptides. Two polypeptides having greater than or equal to about 60% similarity are presumptively homologous.

[0049] "Host," "Host cell" or "host cells" refers to cells expressing a heterologous polynucleotide molecule. The term "heterologous" means non-native. For instance, when a gene that is not normally expressed in an organism is introduced and expressed in that host organism, such an expression is heterologous. Host cells of the present disclosure express polynucleotides encoding KmLATl or PgLAT2 or a fragment thereof. Examples of suitable host cells useful in the present disclosure include, but are not limited to, prokaryotic and eukaryotic cells. Specific examples of such cells include bacteria of the genera Escherichia,

Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus; fungi, particularly filamentous fungi such as Trichoderma and Aspergillus, Phanerochaete chrysosporium and other white rot fungi; also other fungi including Fusaria, molds, and yeast including Saccharomyces sp., Pichia sp., and Candida sp. and the like; plants e.g. Arabidopsis, cotton, barley, tobacco, potato, and aquatic plants and the like; SF9 insect cells (Summers and Smith, 1987, Texas Agriculture Experiment Station Bulletin, 1555), and the like. Other specific examples include mammalian cells such as human embryonic kidney cells (293 cells), Chinese hamster ovary (CHO) cells (Puck et al., 1958, Proc. Natl. Acad. ScL USA 60, 1275-1281), human cervical carcinoma cells (HELA) (ATCC CCL T), human liver cells (Hep G2) (ATCC HB8065), human breast cancer cells (MCF-7) (ATCC HTB22), human colon carcinoma cells (DLD-I) (ATCC CCL 221), Daudi cells (ATCC CRL-213), murine myeloma cells such as P3/NSI/l-Ag4-l (ATCC TIB-18), P3X63Ag8 (ATCC TIB-9), SP2/0-Agl4 (ATCC CRL-1581) and the like. The most preferred host is Saccharomyces cerevisiae.

[0050] "Hybridization" refers to the pairing of complementary polynucleotides during an annealing period. The strength of hybridization between two polynucleotide molecules is impacted by the homology between the two molecules, stringency of the conditions involved, the melting temperature of the formed hybrid and the G:C ratio within the polynucleotides. [0051] "Identity" refers to a comparison of two different DNA or protein sequences by comparing pairs of nucleic acid or amino acids within the two sequences. Methods for determining sequence identity are known. See, for example, computer programs commonly employed for this purpose, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wisconsin), that uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math, 2: 482- 489.

[0052] "Isolated" refers to a polynucleotide or polypeptide that has been separated from at least one contaminant (polynucleotide or polypeptide) with which it is normally associated. For example, an isolated polynucleotide or polypeptide is in a context or in a form that is different from that in which it is found in nature. [0053] "Nucleic acid sequence" refers to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide chain. The deoxyribonucleotide sequence thus codes for the amino acid sequence.

[0054] "Polynucleotide" refers to a linear sequence of nucleotides. The nucleotides may be ribonucleotides, or deoxyribonucleotides, or a mixture of both. Examples of polynucleotides in this context include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. The polynucleotides may contain one or more modified nucleotides. [0055] "Protein," "peptide," and "polypeptide" are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers. [0056] "Purify," or "purified" refers to a target protein makes up for at least about

90% of a composition. In other words, it refers to a target protein that is free from at least 5- 10% of contaminating proteins. Purification of a protein from contaminating proteins may be accomplished using known techniques, including ammonium sulfate or ethanol precipitation, acid precipitation, heat precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, size-exclusion chromatography, and lectin chromatography. Various protein purification techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates).

[0057] "Selectable marker" refers to a marker that identifies a cell as having undergone a recombinant DNA or RNA event. Selectable markers include, for example, genes that encode antimetabolite resistance such as the DHFR protein that confers resistance to methotrexate (Wigler et al, 1980, Proc Natl Acad Sci USA 77:3567; O'Hare et al., 1981, Proc Natl Acad Sci USA, 78: 1527), the GPT protein that confers resistance to mycophenolic acid (Mulligan & Berg, 1981, PNAS USA, 78:2072), the neomycin resistance marker that confers resistance to the aminoglycoside G-418 (Calberre-Garapin et al., 1981, J MoI Biol, 150: 1), the Hygro protein that confers resistance to hygromycin B (Santerre et al., 1984, Gene 30: 147), and the Zeocin™ resistance marker (Invitrogen). In addition, the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes may be employed in tk ^" , hgprt ^" and aprt ^" cells, respectively. [0058] "Transform" means the process of introducing a gene into a host cell. The gene may be foreign in origin, but the gene may also derive from the host. A transformed host cell is termed a "transformant." The introduced gene may be integrated onto the chromosome of the host, or the gene may remain on a stand-alone vector independent of the host chromosomes.

[0059] "Variant", as used herein, means a polynucleotide or polypeptide molecule that differs from a reference molecule. Variants may include nucleotide changes that result in amino acid substitutions, deletions, fusions, or truncations in the resulting variant polypeptide when compared to the reference polypeptide.

[0060] "Vector," "extra-chromosomal vector" or "expression vector" refers to a first polynucleotide molecule, usually double-stranded, which may have inserted into it a second polynucleotide molecule, for example a foreign or heterologous polynucleotide. The heterologous polynucleotide molecule may or may not be naturally found in the host cell, and may be, for example, one or more additional copy of the heterologous polynucleotide naturally present in the host genome. The vector is adapted for transporting the foreign polynucleotide molecule into a suitable host cell. Once in the host cell, the vector may be capable of integrating into the host cell chromosomes. The vector may optionally contain additional elements for selecting cells containing the integrated polynucleotide molecule as well as elements to promote transcription of mRNA from transfected DNA. Examples of vectors useful in the methods disclosed herein include, but are not limited to, plasmids, bacteriophages, cosmids, retroviruses, and artificial chromosomes. [0061] For purpose of this disclosure, unless otherwise stated, the techniques used may be found in any of several well-known references, such as: Molecular Cloning: A Laboratory Manual (Sambrook et al. (1989) Molecular cloning: A Laboratory Manual), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991 Academic Press, San Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, 3d., (1990) Academic Press, Inc.), PCR Protocols: A Guide to Methods and Applications (Innis et al. (1990) Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2 ^nd ed. (R.I. Freshney (1987) Liss, Inc., New York, NY), and Gene Transfer and Expression Protocols, pp 109-128, ed. EJ. Murray, The Humana Press Inc., Clifton, N.J.).

[0062] Unless otherwise indicated, the term "yeast," "yeast strain" or "yeast cell" refers to baker's yeast, Saccharomyces cerevisiae. Other yeast species, such as Kluyveromyces marxianus or Pichia guilliermondii, are referred to as non-conventional yeast in this disclosure. Yeast strains of S. cerevisiae and plasmids used for this disclosure are listed in Table 1. The yeast Kluyveromyces marxianus CBS- 1089 is obtained from the Centraalbureau voor Schimmel cultures (CBS) collection. Pichia guilliermondii NRRL Y- 2075 is obtained from the Agricultural Research Service Culture Collection (NRRL).

Table 1. S. cerevisiae Strains and Plasmids Used in The Disclosure

Strain Genotype Plasmids

BFY002 MATa urα3-52 trpl-δ63 his3-δ200 Ieu2-δl yhrlO4w::LEU2

BFY507 MATa urα3-52 trpl-A63 his3-A200 Ieu2-Al yhrlO4w::LEU2 pl38,p42 adapted for growth on L-arabinose

BFY518 same as BF Y507 pl38

BFY566 same as BF Y518 pl38,pl71

BF Y590 same as BF Y518 gαl2A : :HIS3 pl38

BFY597 same as BFY590 pl38,p42

BFY598 same as BFY590 pl38, pl87

BFY605 same as BFY590 pl38,p244

BFY604 MATα his3Al leu2A0 urα3A0 metl5A0 gαl80A::G418 trplA

BFY607 Same as BFY604 pl38

BFY609 Same as BFY607 pl2, pl3, pl38

BF Y612 Same as BF Y607 pl2, pl38, p204

BFY625 Same as BFY609, adapted for growth on L-arabinose pl2, pl3, pl38

BFY626 Same as BFY612, adapted for growth on L-arabinose pl2, _P 138, p204

BFY057 MATα his 3Dl leu2D0 urα3D0 met 15D0 gαl80D::G418 yhrlO4w::LEU2

BFY534 same as BFY057 pl44, pl65 BFY535 same as BFY057 pl44, pl3

Plasmid Marker and expressed genes pl2, pl3 URA3 or HIS3 control vectors respectively p42 URA3, GAL2 over-expression pl38 TRPl, B. subtilis αrαA, E. coli αrαB, E. coli αrαD pl71 HIS3, 8.8 kb K. mαrxiαnus genomic DNA fragment pl87 URA3, KmLATl over-expression plasmid p204 HIS3, PgLAT2 over-expression plasmid p244 URA3, YgLAT2 over-expression plasmid pl44 E. coli αrαB,D; B. subtilis αrαA in pBFY012 pl65 HIS3, GAL2 over-expression

[0063] Yeast strains may be grown on liquid or solid media with 2% agar for solid media. Where appropriate, some amino acids or nucleic acids are purposely left out from the media for plasmid maintenance. Growth conditions are typically 30 ⁰ C unless otherwise indicated, with shaking in liquid cultures. An anaerobic condition is generally more favorable to metabolize the various sugars to ethanol.

[0064] Yeast cells may be grown in rich media YPD or minimum media conventionally used in the field. YPD medium contains about 1% yeast extract, 2% peptone and 2% dextrose. Yeast minimum media typically contains 0.67% of yeast nitrogen base ("YNB") without amino acids supplemented with appropriate amino acids or purine or pyrimidine bases. A number of sugar, typically at 2% unless otherwise indicated, may be used as carbon source, including glucose (dextrose), galactose, maltose or L-arabinose among others. Adaptation for growth on L-arabinose is performed as described in Becker and Boles (2003) with modifications as detailed in Example 3.

[0065] Over-expression plasmids are constructed by cloning the gene for over- expression downstream of the S. cerevisiae PGKl or TDH3 promoter in a 2μ-based vector. Other like overexpression plasmid for expression of a gene may also be used as is known in the art. Construction of DNA library is detailed in the Examples. [0066] E. coli cells may be grown in LB liquid media or on LB agar plates supplemented with ampicillin at 100 μg/ml as needed. Transformation of E. coli DH5α is by electrotransformation according to a protocol by Invitrogen (Invitrogen 11319-019). After transformation, the bacterial cells are plated on LB plates containing 100 μg/ml ampicillin for selection. Transformation of S. cerevisiae was performed using a DMSO-enhanced lithium- acetate procedure as described with the following modifications (Hill et al., 1991). Cells are harvested and initially washed in water. 600 μl of PEG4000 solution is added and 70 μl DMSO is added just prior to heat shocking. Cells are heat-shocked for 15 min at 42°C and the last wash step is skipped. Cells are resuspended in 10 mM TE solution and plated. [0067] Yeast DNA is isolated using the Easy DNA kit according to manufacturer's protocol (Invitrogen, Kl 800-01). DNA manipulations and library construction are performed as described in Molecular Cloning: A Laboratory Manual (1989), except otherwise specifically indicated in this disclosure. Plasmids are cured from yeast by growing the strain in rich non-selective media overnight followed by plating on non-selective media. Isolated colonies are replica plated to screen for loss of selective markers. Plasmid rescue is performed by transforming isolated yeast DNA into E. coli followed by isolation and

characterization. E. coli plasmid isolation is accomplished using plasmid spin mini-prep kit according to the manufacturer's manual (Qiagen, 27106). PCR-based chromosomal walking is performed using the Universal Genome Walker Kit as described (BD Biosciences, Kl 807-

1).

[0068] For transport assays, cells may be grown in minimal media supplemented with

20 g/L of L-arabinose. Cells are collected in mid-growth and washed twice before suspension in water at 30 mg/ml. Uptake of L-(l- ¹⁴ C)arabinose (54 mCi/mmol, Moravek Biochemicals Inc.) or D-(I- ¹⁴ C) galactose (57 mCi/mmol, Amersham Biosciences) is measured as previously described by Stambuk et al. (2003). Assays are performed in 30 seconds to maintain initial rates after appropriate experiments to ensure uptake is linear for at least 1 minute. Transport activity is described as nano-moles of labeled sugar transported per mg cell dry weight per minute. Inhibition and competition assays are performed as previously described by Stambuk et al. (2003).

[0069] Embodiments described herein provide systems, tools and methods for the identification of yeast strains efficient in utilization of L-arabinose as a sugar source. In some aspects the identified yeast strains are amenable to genetic and biochemical manipulation. In further aspects certain identified yeast strains are shown to include a single component responsible for transporting arabinose into the yeast. These identified single components correspond to arabinose transporter genes that can then be identified and cloned (as described herein).

[0070] Identification of arabinose utilizing or fermenting yeast strains in accordance with embodiments described herein includes: (1) selecting yeast strains for screening that are not pathogenic, did not, or rarely, form hyphae and grow primarily as a single cell(s); (2) growing selected yeast strain(s) or minimal growth medium containing from 0.2 to 2% L- arabinose as a sole sugar source; (3) classifying the yeast strain for its capacity to utilize L- arabinose (typically via periodic growth measurements of optical density); (4) determining if a strain that shows growth on arabinose is amenable to genetic and biochemical manipulation, i.e., only strains that are amenable to manipulation are useful for ultimate cloning and identification of the arabinose transporter gene; and (5) perform arabinose transport assays on the selected yeast strains that grow on L-arabinose to identify single affinity arabinose transporter strains of yeast. Single component high affinity arabinose transporter systems are targeted for further identification of arabinose transporter genes (see below). As discussed in detail in Example 1, at least two strains of yeast were identified as containing high affinity, single component transporter systems, K. marxianus and P. guilliermondii. As detailed

below and in Examples 2-9, the genes identified are KmLATI and PgLAT2, other like genes are identified able using the novel methods and tools described herein, each of which is within the scope of this disclosure.

[0071] Briefly, and by way of example, the KmLATl transporter gene was identified using complementation of a strain adapted for growth on L-arabinose as described above. This strain was able to utilize L-arabinose only if a suitable transporter was present. After adaptation, the plasmid carrying the GAL2 transporter was cured (removed) from the strain rendering this strain unable to grow on L-arabinose. A genomic library was then introduced and colonies selected that regained the ability to grow on L-arabinose. The genomic fragment isolated in this manner contained the ORF for the KmLATl transporter. [0072] The PgLAT2 transporter gene was identified using differential display

(differential expression). The identified strain using methods described herein was separately grown on L-arabinose and a control sugar (D-xylose). Total RNA was isolated from cells grown on the two sugars and analyzed to detect MRNAs that were only expressed or much more highly expressed when the cells were grown on L-arabinose compared to the samples from the MRNA from cells grown on the control sugar. The gene fragments corresponding to the differentially expressed genes were sequenced and the complete gene was then isolated from the genome of P. guilliermondii by genome walking using oligonucleotide primers designed from the sequences of the fragments. Complete sequence of the gene was then determined and the gene was engineered for expression in S. cerevisiae. [0073] Note that similar techniques can be used to identify transporter genes from other like L-arabinose fermenting yeast stems. Examples 1-10 illustrate various of the methods described herein.

[0074] Sequencing results showed that the KmLATl gene contains an open reading frame ("ORF") of 1668 bp in length. The predicted amino acid sequence of KmLatl shares homology with high-affinity glucose transporters, in particular, with HGTl from K lactis (Table 2). KmLatl transporter shows a much higher sequence similarity with high-affinity glucose transporters from non-conventional yeast than with transporter proteins encoded by the bacterial araE gene or hexose transporters from S. cerevisiae (See Fig. 8).

Table 2. Properties and similarities of KmLatl to other sugar transporters.

gene Predicted pi of Predicted Degree of Organism Putative function protein protein transmemidentity (%) of gene product

(no. of brane /similarity (%) aa/no. of regions kDa)

KmLATl 556 / 61.3 8.22 12 - L-arabmose transporter

K marxianus

KlHGTl 551 / 60.8 5.76 12 77 / 89 high affinity glucose

K lactis ² transporter

AEL042Cp 547 / 59.8 8.82 12 65 / 82 A gossypu ³ putative hexose transporter

DEHA0E01738 545 / 61.1 5.55 12 52 / 70 D hansenif hexose g transporter

CaHGTl 545 / 60.7 8.05 12-13 50 / 71 C albicans ⁵ putative hexose transporter

CaHGT2 545 / 60.4 8.48 12-14 51 / 71 C albicans ⁶ putative hexose transporter

Accession numbers: 1 : Not yet assigned, 2: 1346290, 3 AEL042C, 4 DEHA0E01738g, 5: CAA76406, 6: orfl9.3668

[0075] Transmembrane regions predicted for KmLatl and PgLat2 by the software

Tmpred show 12 transmembrane regions with a larger intercellular loop between regions 6 and 7 (Fig. 2) (See Hofmann et al, 1993), typical of Gal2 and other yeast sugar transporters having 10-12 transmembrane regions {See e.g., Alves-Araujo et al., 2004; Day et al., 2002; Kruckeberg et al., 1996; Pina et al. ,2004; and Weierstall et al. 1999). [0076] Like other members of the transporter family, and in particular sugar transporters, KmLatl and PgLat2 polypeptides are useful in facilitating the uptake of various sugar molecules into the cells. It is envisioned that KmLatl or PgLat2 polypeptides may be used for other purposes, for example, in analytical instruments or other processes where uptake of sugar is required. KmLatl or PgLat2 polypeptides may be used alone or in combination with one or more other transporters to facilitate the movement of molecules across a membrane structure, which function may be modified by one skilled in the relevant art, all of which are within the scope of the present disclosure.

[0077] KmLatl polypeptides may include isolated polypeptides having an amino acid sequence as shown below in Example 2; and in SEQ ID NO:2, as well as variants and derivatives, including fragments, having substantial sequence similarity to the amino acid sequence of SEQ ID NO:2 and that retain any of the functional activities of KmLatl. PgLat2 polypeptides may include isolated polypeptides having an amino acid sequence as shown

below in Example 5; and in SEQ ID NO:4, as well as variants and derivatives, including fragments, having substantial sequence similarity to the amino acid sequence of SEQ ID NO:4 and that retain any of the functional activities of PgLat2. The functional activities of the KmLatl or PgLat2 polypeptides include but are not limited to transport of L-arabinose across cell membrane. Such activities may be determined, for example, by subjecting the variant, derivative, or fragment to a arabinose transport assay as detailed in Example 4. [0078] Variants and derivatives of KmLatl or PgLat2 include, for example, KmLatl or PgLat2 polypeptides modified by covalent or aggregative conjugation with other chemical moieties, such as glycosyl groups, polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups, and the like.

[0079] The amino acid sequence of these KmLatl or PgLat2 variants or derivatives is preferably at least about 60% identical, more preferably at least about 70% identical, still more preferably at least 80% identical, or in some embodiments at least about 90%, 95%, 96%, 97%, 98%, or 99% identical, to the KmLatl and PgLat2 amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The percentage sequence identity, also termed homology (see definition above) may be readily determined, for example, by comparing the two polypeptide sequences using any of the computer programs commonly employed for this purpose, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wisconsin), which uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482-489. [0080] Variants and derivatives of the KmLatl or PgLat2 polypeptides may further include, for example, fusion proteins formed of a KmLatl or PgLat2 polypeptide and another polypeptide. Fusion protein may be formed between a fragment of the KmLatl or PgLat2 polypeptide and another polypeptide, such that the fusion protein may retain all or only part of the activities normally performed by the full-length KmLatl or PgLat2 polypeptide. Preferred polypeptides for constructing the fusion protein include those that facilitate purification or oligomerization, or those that enhance KmLatl or PgLat2 stability and/or transport capacity or transport rate for sugars, especially for arabinose. Preferred polypeptides may also include those that gain enhanced transport capability when fused with KmLatl, PgLat2 or fragments thereof.

[0081] KmLatl or PgLat2 variants and derivatives may contain conservatively substituted amino acids, meaning that one or more amino acid may be replaced by an amino acid that does not alter the secondary and/or tertiary structure of the polypeptide. Such substitutions may include the replacement of an amino acid, by a residue having similar

physicochemical properties, such as substituting one aliphatic residue (He, VaI, Leu, or Ala) for another, or substitutions between basic residues Lys and Arg, acidic residues GIu and Asp, amide residues GIn and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr. Phenotypically silent amino acid exchanges are described more fully in Bowie et al., 1990. In addition, functional KmLatl or PgLat2 polypeptide variants include those having amino acid substitutions, deletions, or additions to the amino acid sequence outside functional regions of the protein.

[0082] The KmLatl or PgLat2 polypeptides may be provided in an isolated form, or in a substantially purified form. The polypeptides may be recovered and purified from recombinant cell cultures by known methods, including, for example, ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Preferably, protein chromatography is employed for purification.

[0083] A preferred form of KmLatl or PgLat2 polypeptides is that of recombinant polypeptides expressed by suitable hosts. In one preferred embodiment, when heterologous expression of KmLatl or PgLat2 is desired, the coding sequences of KmLatl or PgLat2 may be modified in accordance with the codon usage of the host. Such modification may result in increase protein expression of a foreign in the host. Furthermore, the hosts may simultaneously produce other transporters such that multiple transporters are expressed in the same cell, wherein the different transporters may form oligomers to transport the same sugar. Alternatively, the different transporters may function independently to transport different sugars. Such recombinant cells may be useful in crude fermentation processing or in other industrial processing.

[0084] KmLatl or PgLat2 polypeptides may be fused to heterologous polypeptides to facilitate purification. Many available heterologous peptides (peptide tags) allow selective binding of the fusion protein to a binding partner. Non-limiting examples of peptide tags include 6-His, thioredoxin, hemaglutinin, GST, and the OmpA signal sequence tag. A binding partner that recognizes and binds to the heterologous peptide may be any molecule or compound, including metal ions (for example, metal affinity columns), antibodies, antibody fragments, or any protein or peptide that preferentially binds the heterologous peptide to permit purification of the fusion protein.

[0085] KmLatl or PgLat2 polypeptides may be modified to facilitate formation of

KmLatl or PgLat2 oligomers. For example, KmLatl polypeptides may be fused to peptide

moieties that promote oligomerization, such as leucine zippers and certain antibody fragment polypeptides, for example, Fc polypeptides. Techniques for preparing these fusion proteins are known, and are described, for example, in WO 99/31241 and in Cosman et.al, 2001. Fusion to an Fc polypeptide offers the additional advantage of facilitating purification by affinity chromatography over Protein A or Protein G columns. Fusion to a leucine-zipper (LZ), for example, a repetitive heptad repeat, often with four or five leucine residues interspersed with other amino acids, is described in Landschultz et al, 1988. [0086] It is also envisioned that an expanded set of variants and derivatives of

KmLATl or PgLAT2 polynucleotides and/or polypeptides may be generated to select for useful molecules, where such expansion is achieved not only by conventional methods such as site-directed mutagenesis but also by more modern techniques, either independently or in combination.

[0087] Site-directed-mutagenesis is considered an informational approach to protein engineering and may rely on high-resolution crystallographic structures of target proteins for specific amino acid changes (van den Burg et al. 1998). For example, modification of the amino acid sequence of KmLatl or PgLat2 polypeptides may be accomplished as is known in the art, such as by introducing mutations at particular locations by oligonucleotide-directed mutagenesis. Site-directed-mutagenesis may also take advantage of the recent advent of computational methods for identifying site-specific changes for a variety of protein engineering objectives (Hellinga, 1998).

[0088] The more modern techniques include, but are not limited to, non-informational mutagenesis techniques (referred to generically as "directed evolution"). Directed evolution, in conjunction with high-throughput screening, allows testing of statistically meaningful variations in protein conformation (Arnold, 1998). Directed evolution technology may include diversification methods similar to that described by Crameri et al. (1998), site- saturation mutagenesis, staggered extension process (StEP) (Zhao et al., 1998), and DNA synthesis/reassembly (U.S. Patent 5,965,408).

[0089] Fragments of the KmLatl or PgLat2 polypeptide may be used, for example, to generate specific anti-KmLatl or PgLat2 antibodies. Using known selection techniques, specific epitopes may be selected and used to generate monoclonal or polyclonal antibodies. Such antibodies have utility in the assay of KmLatl or PgLat2 activity as well as in purifying recombinant KmLatl or PgLat2 polypeptides from genetically engineered host cells. [0090] The disclosure also provides polynucleotide molecules encoding the KmLatl or PgLat2 polypeptides discussed above. KmLATl or PgLAT2 polynucleotide molecules of

the disclosure include polynucleotide molecules having the nucleic acid sequence shown in SEQ ID NO: 1 and SEQ ID NO:3, respectively; polynucleotide molecules that hybridize to the nucleic acid sequence of SEQ ID NO: 1 and SEQ ID NO:3, respectively, under high stringency hybridization conditions (for example, 42°, 2.5 hr., 6X SCC, 0.1%SDS); and polynucleotide molecules having substantial nucleic acid sequence identity with the nucleic acid sequence of SEQ ID NO: 1 and SEQ ID NO:3, respectively. [0091] The KmLATl or PgL AT2 polynucleotide molecules of the disclosure are preferably isolated molecules encoding the KmLatl or PgLat2 polypeptide having an amino acid sequence as shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, as well as derivatives, variants, and useful fragments of the KmLATl or PgLAT2 polynucleotide. The KmLATl or PgL AT2 polynucleotide sequence may include deletions, substitutions, or additions to the nucleic acid sequence of SEQ ID NO: 1 and SEQ ID NO:3, respectively. [0092] The KmLATl or PgLAT2 polynucleotide molecule of the disclosure may be cDNA, chemically synthesized DNA, DNA amplified by PCR, RNA, or combinations thereof. Due to the degeneracy of the genetic code, two DNA sequences may differ and yet encode identical amino acid sequences. The present disclosure thus provides an isolated polynucleotide molecule having a KmLATl or PgLAT2 nucleic acid sequence encoding KmLatl or PgLat2 polypeptide, wherein the nucleic acid sequence encodes a polypeptide having the complete amino acid sequences as shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, or variants, derivatives, and fragments thereof.

[0093] The KmLATl or PgLAT2 polynucleotides of the disclosure have a nucleic acid sequence that is at least about 60% identical to the nucleic acid sequence shown in SEQ ID NO: 1 and SEQ ID NO:3, respectively, in some embodiments at least about 70% identical to the nucleic acid sequence shown in SEQ ID NO: 1 and SEQ ID NO:3, respectively, in other embodiments at least about 80% identical to the nucleic acid sequence shown in SEQ ID NO: 1 and SEQ ID NO:3, respectively and in other embodiments at least about 90%, 95%, 96%, 97%, 98%, 99%, identical to the nucleic and sequence shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Nucleic acid sequence identity is determined by known methods, for example by aligning two sequences in a software program such as the BLAST program (Altschul, S.F et al. (1990) J. MoI. Biol. 215:403-410, from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). [0094] The KmLATl or PgL AT2 polynucleotide molecules of the disclosure also include isolated polynucleotide molecules having a nucleic acid sequence that hybridizes under high stringency conditions (as defined above) to a the nucleic acid sequence shown in

SEQ ID NO: 1 and SEQ ID NO:3, respectively. Hybridization of the polynucleotide is to at least about 15 contiguous nucleotides, or at least about 20 contiguous nucleotides, and in other embodiments at least about 30 contiguous nucleotides, and in still other embodiments at least about 100 contiguous nucleotides of the nucleic acid sequence shown in SEQ ID NO: 1 and SEQ ID NO:3, respectively.

[0095] Useful fragments of the KmLATl or PgLAT2 polynucleotide molecules described herein, include probes and primers. Such probes and primers may be used, for example, in PCR methods to amplify and detect the presence of KmLATl or PgLAT2 polynucleotides in vitro, as well as in Southern and Northern blots for analysis of KmLATl or PgLAT2. Cells expressing the KmLATl or PgLAT2 polynucleotide molecules may also be identified by the use of such probes. Methods for the production and use of such primers and probes are known. For PCR, 5' and 3' primers corresponding to a region at the termini of the KmLATl or PgLAT2 polynucleotide molecule may be employed to isolate and amplify the KmLATl or PgLAT2 polynucleotide using conventional techniques. [0096] Other useful fragments of the KmLATl or PgL AT2 polynucleotides include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence capable of binding to a target KmLATl or PgLAT2 mRNA (using a sense strand), or DNA (using an antisense strand) sequence.

[0097] The present disclosure also provides vectors containing the polynucleotide molecules, as well as host cells transformed with such vectors. Any of the polynucleotide molecules of the disclosure may be contained in a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. The vectors may further include suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, fungal, bacterial, viral, or insect genes, operably linked to the KmLATl or PgLAT2 polynucleotide molecule. Examples of such regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the target protein. Thus, a promoter nucleotide sequence is operably linked to a KmLATl or PgLAT2 DNA sequence if the promoter nucleotide sequence directs the transcription of the KmLATl or PgLAT2 sequence.

[0098] Selection of suitable vectors for the cloning of KmLATl or PgLAT2 polynucleotide molecules encoding the target KmLatl or PgLat2 polypeptides of this disclosure will depend upon the host cell in which the vector will be transformed, and, where

applicable, the host cell from which the target polypeptide is to be expressed. Suitable host cells for expression of KmLatl or PgLat2 polypeptides include prokaryotes, yeast, and higher eukaryotic cells, each of which is discussed below.

[0099] The KmLatl or PgLat2 polypeptides to be expressed in such host cells may also be fusion proteins that include sequences from other proteins. As discussed above, such regions may be included to allow, for example, enhanced functionality, improved stability, or facilitated purification of the KmLatl or PgLat2 polypeptide. For example, a nucleic acid sequence encoding a peptide that binds strongly to arabinose may be fused in- frame to the transmembrane sequence of the KmLatl or PgLat2 polypeptides so that the resulting fusion protein binds arabinose and transports the sugar across the cell membrane at a higher rate than the KmLatl or PgLat2 transporter.

[00100] Suitable host cells for expression of target polypeptides include prokaryotes, yeast, and higher eukaryotic cells. Suitable prokaryotic hosts to be used for the expression of these polypeptides include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus. [00101] Expression vectors for use in prokaryotic hosts generally comprise one or more phenotypic selectable marker genes. Such genes encode, for example, a protein that confers antibiotic resistance or that supplies an auxotrophic requirement. A wide variety of such vectors are readily available from commercial sources. Examples include pSPORT vectors, pGEM vectors (Promega, Madison, WI), pPROEX vectors (LTI, Bethesda, MD), Bluescript vectors (Stratagene), and pQE vectors (Qiagen).

[00102] KmLATl or PgLAT2 may also be expressed in yeast host cells from genera including Saccharomyces, Pichia, and Kluveromyces. Preferred yeast host is S. cerevisiae. Yeast vectors will often contain an origin of replication sequence from a 2μ yeast plasmid for high copy vectors and a CEN sequence for a low copy number vector. Other sequences on a yeast vector may include an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Vectors replicable in both yeast and E. coli (termed shuttle vectors) are preferred. In addition to the above-mentioned features of yeast vectors, a shuttle vector will also include sequences for replication and selection in E. coli.

[00103] Insect host cell culture systems may also be used for the expression of

KmLatl or PgLat2 polypeptides. The target polypeptides are preferably expressed using a baculovirus expression system, as described, for example, in the review by Luckow and Summers, 1988.

[00104] The choice of a suitable expression vector for expression of KmLatl or

PgLat2 polypeptides will depend upon the host cell to be used. Examples of suitable expression vectors for E. coli include pET, pUC, and similar vectors as is known in the art. Preferred vectors for expression of the KmLatl or PgLat2 polypeptides include the shuttle plasmid pIJ702 for Streptomyces lividans, pGAPZalpha-A, B, C and pPICZalpha-A, B, C (Invitrogen) for P 'ichia pastor is, and pFE-1 and pFE-2 for filamentous fungi and similar vectors as is known in the art. The vectors preferred by expression in S. cerevisiae are listed in Table 1.

[00105] Modification of a KmLATl or PgLAT2 polynucleotide molecule to facilitate insertion into a particular vector (for example, by modifying restriction sites), ease of use in a particular expression system or host (for example, using preferred host codons), and the like, are known and are contemplated for use as descibed herein. Genetic engineering methods for the production of KmLatl or PgLat2 polypeptides include the expression of the polynucleotide molecules in cell free expression systems, in host cells, in tissues, and in animal models, according to known methods.

[00106] This disclosure also provides reagents, compositions, and methods that are useful for analysis of KmLatl or PgLat2 activity and for assessing the amount and rate of arabinose transport.

[00107] The KmLatl or PgLat2 polypeptides of the present disclosure, in whole or in part, may be used to raise polyclonal and monoclonal antibodies that are useful in purifying KmLatl or PgLat2, or detecting KmLatl or PgLat2 polypeptide expression, as well as a reagent tool for characterizing the molecular actions of the KmLatl or PgLat2 polypeptide. Preferably, a peptide containing a unique epitope of the KmLatl or PgLat2 polypeptide is used in preparation of antibodies, using conventional techniques. Methods for the selection of peptide epitopes and production of antibodies are known. See, for example, Antibodies: A Laboratory Manual, Harlow and Land (eds.), 1988 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), 1980 Plenum Press, New York. [00108] Agents that modify, for example, increase or decrease, KmLatl or PgLat2 transport of arabinose or other sugars may be identified, for example, by the transport assay described in Example 5. Performing the transport assay in the presence or absence of a test agent permits screening of such agents.

[00109] The KmLatl or PgLat2 transport activity is determined in the presence or absence of a test agent and then compared. For instance, a lower KmLatl transport activity

in the presence of the test agent, than in the absence of the test agent, indicates that the test agent has decreased the activity of the KmLatl. Stimulators and inhibitors of KmLatl or PgLat2 may be used to augment, inhibit, or modify KmLatl or PgLat2 transport activity, and therefore may have potential industrial uses as well as potential use in further elucidation of the molecular actions of KmLatl or PgLat2.

[00110] The KmLatl or PgLat2 polypeptide of the disclosure is an effective arabinose transporter. In the methods of the disclosure, the sugar transporting effects of KmLatl or PgLat2 are achieved by mixing cells expressing KmLatl or PgLat2 with pure sugar or sugar- containing biomass. KmLatl or PgLat2 may also be used in a cell-free system. KmLatl or PgLat2 may be used under other conditions, for example, at elevated temperatures or under acidic pH. Other methods of using KmLatl or PgLat2 to transport sugar, especially arabinose, for fermentation, are envisioned to be within the scope of the present disclosure. KmLatl or PgLat2 polypeptides may be used in any known application currently utilizing a sugar transporter, all of which are within the scope of the disclosure. It should be noted that the KmLatl and PgLat2 polypeptides are also capable of transporting other sugars, including D-xylose, and thus may have utility for transport of other biomass-derived sugars. [00111] It is also shown in this disclosure that Gal2p is an effective L-arabinose transporter at high concentrations of arabinose whereas KmLatl or PgLat2 may be more effective at different concentrations of L-arabinose. Combination of the Gal2p and the two new transporters from non-conventional yeast may be employed to provide complementary transport into S. cerevisiae of L-arabinose down to very low residual concentration of arabinose.

[00112] It is shown that combinatorial expression of Gal2p and PgLat2 may enhance the overall rate and extent of arabinose utilization by recombinant S. cerevisiae cells expressing these transporters. As shown in Example 9, the doubling time for S. cerevisiae strain expressing both PgLat2 and Gal2p is shorter than S. cerevisiae cells expressing Gal2p alone (15 hours vs. 19 hours), suggesting that L-arabinose uptake may be enhanced by the synergistic effect of PgLat2 and Gal2p in these cells. Moreover, the PgLat2 expressing strain appears to grow to a higher overall optical density at saturation, suggesting that this strain was able to utilize the carbon source (L-arabinose) in the medium more completely. This hypothesis is supported by HPLC analysis of the final culture media (Table 3) which indicates that there is significantly less residual L-arabinose in the culture of cells expressing Gal2p and PgLat2 than in the culture of those expressing Gal2p alone. Thus, heterologous expression of either or both KmLatl and PgLat2 in S. cerevisiae may enhance arabinose

utilization by facilitating arabinose transport when the concentration of arabinose is relatively low.

Table 3. Doubling times and HPLC Measurement of Residual Arabinose Concentration

*starting L-arabinose concentration 1.89 g/L and media without L-arabinose had an undetectable level (<0.1 g/L).

[00113] Note that yeast strains BFY013, BFY534, BFY598 and BFY626 were deposited by the Inventors at American Type Culture Collection, 10801 University Boulevard, Manassas, VA, on March 16, 2007. Strain BFYO 13 has accession number strain BFY504 has accession number ; strain BFY598 has accession number and strain BFY626 has accession number . All strains were deposited in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Protection.

[00114] The examples herein illustrate the disclosure and are not meant as limiting in nature. The chemicals, biological agents and other ingredients are presented as typical components or reactants, and the procedures described herein may represent but one of the typical ways to accomplish the goal of the particular experiment. It is understood that various modification may be derived in view of the foregoing disclosure without departing from the spirit of the present disclosure.

EXAMPLES Example 1-Identification of Yeast Strains Capable of Efficiently Fermenting Arabinose

[00115] To better understand L-arabinose transport and use in yeasts and to identify a source for efficient L-arabinose transporters, 165 non-Saccharomyces yeast strains were

studied. These yeast strains were arranged into 6 groups based on the minimum time required to utilize 20 g/L of L-arabinose. Transport rates of L-arabinose were determined for several strains and a more comprehensive transport studies was done in four selected strains. Detailed transport kinetics in Arxula adeninivorans showed a transport system consisting of low and high affinity components while that of Debaryomyces hansenii var.fabryii, Kluyveromyces marxianus and Pichia guilliermondii showed that these strains have single component, high affinity active transport systems.

[00116] The rationale for selecting yeast species and strains was based on reasoning that an organism that grows well on L-arabinose must have an efficient mechanism for the uptake of this sugar. In order to facilitate future experiments, we limited our search for species that were not reported to be pathogenic, did not, or rarely, formed hyphae, and grew primarily as single cells. In order to choose strains based on these criteria, we relied, in part, on the publication on yeasts by Barnett, Payne and Yarrow (Barnett, et al, 2000), previously described L-arabinose fermenting strains (Dien, et al., 1996; Kurtzman and Dien, 1998), and descriptions in the publications by various culture collections. We obtained 165 strains from 123 different species from the American Type Culture Collection (ATCC), the National Center for Agricultural Utilization Research (NRRL), or the Centraalbureau voor Schimmelculrures (CBS) (Table I).

[00117] The routine growth medium for the growth and maintenance of the yeast strains was YPD (1% Bacto-yeast extract (Difco), 2% Bacto-peptone (Difco), 2% dextrose, and when needed for solidification, 2 % Bacto agar (Difco)). For the testing of growth on L- arabinose, similar medium (YPA) was used in which glucose was replaced with L-arabinose. Minimal medium contained 0.67% yeast nitrogen base without amino acids and 2% glucose or 2% L-arabinose. In order to determine auxotrophic requirements of various mutants, "drop-out" media (Sigma Aldrich or Clontech) were used. These media contain various supplements, but are missing adenine, uracil, or one of the amino acids. [00118] Cells, grown in YDP medium to early stationary phase, were collected by centrifugation, washed with water and suspended in 25 ml YPA medium at an initial density of ODδoo of 0.2 /ml. Cultures were incubated at 30 ⁰ C with shaking at 220 rpm. Growth was monitored by periodic measurement of the optical density of the cultures. Utilization of L- arabinose was determined by measuring the sugar remaining in the medium after various periods of growth by the analysis of the filtered media by high pressure liquid chromatography (HPLC) on Hewlett-Packard (HP) 1090 instrument using a Bio-Rad HPX-

87H hydrogen ion resin column and an HP 1047 A external refractive index detector. The mobile phase, 0.001 N H ₂ SO ₄ , was run at 55°C with a flow rate of 0.6 ml/min. [00119] Cells were grown on L-arabinose to mid-log phase and collected by centrifugation, washed twice in water, and then suspended in water. The yeast suspension was adjusted to 30-60 mg dry weight/ml. Uptake rates and inhibition of L-arabinose transport were measured as previously described (Stambuk, et al., 2003). For our initial transport studies, we had purchased and used L-(l- ¹⁴ C)arabinose from American Radiolabeled Chemicals, Inc. (St Louis, MO. USA). However, we discovered that the l- ¹⁴ C-labeled L- arabinose from this source was heavily contaminated with other radioactive compounds (see below), transport experiments reported here were done using L-(l- ¹⁴ C)arabinose (54 mCi/mmol) that was custom-synthesized by Moravek Biochemicals Inc. (Brea, CA. USA). The analysis of the radioactive substrate was performed by thin layer chromatography (TLC) as described (Han and Robyt, 1998) using Whatman Silica Gel 6θA TLC plates (cat# 4410- 221) for the separation of the compounds to visualize the products of this reaction. We also established that the compound migrating to the position expected for L-arabinose is, in fact, L-arabinose. For these determinations we used a previously described method (Sturgeon, 1986). Briefly, the preparations were dried down and resuspended to a final concentration of 180 μM in a 50 μL volume containing 80 mM Tris HCl, pH 8.6 and 500 μM NAD. This suspension was then treated with β-galactose dehydrogenase (Roche) for one hour at room temperature. Disappearance of L-arabinose, which is oxidized to form L-arabinono-1,5- lactone, was confirmed by analyzing the reaction mixture by the TLC method described above.

[00120] Mutagenesis. Cells were grown in YPD to stationary phase, washed, and then treated with 3% ethyl methanesulfonate (EMS) in 0.1 M potassium phosphate buffer (pH 7.2) for 1.0 h or 1.5 h at 30 ⁰ C. At the end of the treatment, EMS was inactivated by diluting the cells in 5% sodium thiosulfate solution. The treated cells were washed and plated on YPD medium. After 2 days of growth, the yeast colonies were tested for auxotrophy by replica plating onto the synthetic minimal medium. For testing of growth of mutants on other sugars, the glucose from the minimal medium was replaced with appropriate sugars.

RESULTS

[00121] Utilization of L-arabinose. The first step in the selection of strains for transport studies was to determine how well various strains utilized L-arabinose for growth.

The efficiency with which the selected strains utilized the sugar was determined by measuring their growth on L-arabinose and by measuring the amount of L-arabinose that was consumed by these strains during various periods of growth. The amount of L-arabinose utilized was determined by HPLC analysis of the growth medium. Based on the rate of L- arabinose utilization, the 165 strains were classified into one of six groups (Tables 4 and 5). The group classification and final optical density of the cultures when grown on L-arabinose are indicated in Table 4 and a summary is provided in Table 5. Further studies of L-arabinose transport focused on the strains in groups 1-3.

Table 4: Yeast strains

Species Strain Group OD ₆₀₀

Ambrosiozyma monospora CBS-2554 2 18.1

Ambrosiozyma monospora* NRRL Y- 1484 2 25.5

Arxula adeninivorans CBS-8244 1 37.2

Bullera coprosmaensis CBS-8284 3 20.9

Bullera dendrophila ATCC-24608 4 4.2

Bullera globispora CBS-6981 4 23.5

Bullera mrakii CBS-8288 3 32.5

Bullera penniseticola CBS-8623 4 16.3

Bullera pseudoalba CBS-7227 3 26.8

Bullera sinensis CBS-7345 5 7.1

Bullera varaiabilis CBS-7354 3 21.6

Bulleromyces albus ATCC- 18568 6 0.3

Bulleromyces albus CBS-6302 4 21.2

Candida arabinofermentans * NRRL YB- 1299 2 34.6

Candida arabinofermentans * NRRL YB- 1984 2 31.3

Candida arabinofermentans NRRL YB-2248 3 26.2

Candida atlantica CBS-5263 3 28.8

Candida auringiensis CBS-6913 3 32.4

Candida auringiensis NRRL Y- 11848 3 34.6

Candida auringiensis NRRL Y- 11849 3 32.5

Candida bertae ATCC-58889 4 32.3

Candida bertae var. bertae CBS-4605 4 32.2

Candida blankii ATCC- 18735 2 37.7

Candida blankii CBS-6734 6 9.7

Candida cellulolytica CBS-7920 2 32.4

Candida chilensis CBS-5719 6 0.2

Candida conglobata CBS-5808 4 24.5

Candida conglobata NRRL Y-1504 3 26.4

Candida diddensiae CBS-6032 3 29.5

Candida diddensiae * NRRL Y-7589 2 26.5

Candida entomaea * NRRL Y-7785 2 33.5

Candida insectorum ATCC-22940 2 35.3

Candida ishiwadae ATCC-22018 6 5.0

Candida ishiwadae CBS-7348 6 8.2

Candida membranifaciens CBS- 1952 2 35.5

Candida membranifaciens * NRRL Y-2089 2 27.1

Candida methanosorbosa CBS-7029 1 35.6

Candida mogii CBS-2032 3 23.6

Candida nanaspora CBS-7200 3 20.8

Candida nitratophila CBS-2027 3 25.6

Candida ovalis * NRRL Y- 17662 2 14.8

Candida peltata CBS-5576 1 32.1

Candida pignaliae CBS-6071 3 23.4

Candida populi CBS-7351 4 26.0

Candida psychrophila CBS-5956 6 0.2

Candida rhagii** ATCC-22983 5 12.3

Candida rhagii CBS-4432 6 5.1

Candida santjacobensis CBS-8183 6 3.8

Candida sequanensis CBS-8118 6 3.9

Candida shehatae var. shehatae CBS-2779 6 3.4

Candida shehatae var. shehatae NRRL Y- 17029 6 4.8

Candida silvicultrix CBS-6269 5 8.0

Candida sonorensis CBS-6793 2 31.1

Candida succiphila CBS-7297 2 32.0

Candida succiphila * NRRL Y- 11997 2 14.4

Candida succiphila NRRL Y- 11998 3 15.4

Candida tenuis CBS-2885 3 25.3

Candida tenuis * * ATCC-10573 5 10.6

Candida vanderwaltii CBS-5524 1 34.8

Cryptococcus albidus ATCC- 10666 5 13.1

Cryptococcus albidus var. albidus CBS-8395 3 32.8

Cryptococcus aerius CBS- 155 3 23.5

Cryptococcus cellulolyticus CBS-8294 3 27.2

Cryptococcus curvatus ATCC- 10567 6 10.3

Cryptococcus curvatus CBS-5324 6 10.4

Cryptococcus luteolus ATCC-32044 6 2.0

Cryptococcus luteolus CBS-8014 2 33.8

Cryptococcus terreus ATCC-11799 4 16.5

Cryptococcus terreus CBS-7528 3 21.5

Cystofilobasidium capitatum CBS-7420 6 0.2

Debaryomyces hansenii** ATCC-2357 5 10.0

Debaryomyces hansenii ATCC-36239 4 21.1

Debaryomyces hansenii CBS-941 3 32.2

Debaryomyces hansenii var.fabryii CBS-2753 1 45.2

Debaryomyces nepalensis NRRL Y-7108 1 25.8

Debaryomyces polymorphus NRRL Y-2022 6 6.4

Debaryomyces robertsiae CBS-2934 5 10

Debaryomyces yamadae NRRL Y-11714 6 5.6

Fellomyces borneensis CBS-8282 5 19.7

Fellomyces chinensis CBS-8278 6 2.8

Fellomyces distylii CBS-8545 4 32.2

Fellomyces fuzhouensis CBS-6133 4 21.8

Fellomyces horovitziae CBS-7515 6 0.2

Fellomyces lichenicola CBS-8315 6 1.7

Fellomyces ogasawarensis CBS-8544 2 37.0

Fellomyces penicillatus ATCC-32128 5 12.4

Fellomyces penicillatus CBS-5491 5 20.0

Fellomyces polyborus ATCC-32821 6 11.5

Fellomyces polyborus CBS-8333 5 18.3

Fellomyces sichuanensis CBS-8277 6 6.4

Fibulobasidium inconspicuum CBS-6963 3 27.7

Filobasidium floriforme ATCC-22367 3 15.2

Filobasidium floriforme CBS-6241 4 14.1

Hansenula glucozyma ATCC- 18938 6 5.4

Kluveromyces marxianus CBS-712 3 20.9

Kluveromyces marxianus CBS- 1089 2 23

Kluveromyces marxianus CBS-1555 2 24.9

Kluveromyces marxianus CBS-1557 3 26.8

Kluveromyces marxianus CBS-2173 3 19.9

Kluveromyces marxianus NRRL Y-8281 3 13.7

Kockovaella machilophila CBS-8607 5 12.7

Kockovaella sacchari CBS-8624 4 21.4

Myxozyma geophila CBS-7219 4 24.9

Myxozyma kluyveri CBS-7332 6 2.5

Myxozyma lipomycoides CBS-7038 3 16.3

Myxozyma melibiosi CBS-2102 3 27.4

Myxozyma monticola CBS-7806 4 13.9

Myxozyma mucilagina CBS-7071 3 17.0

Myxozyma neglecta CBS-7058 3 20.2

Myxozyma vanderwaltii CBS-7517 3 26.4

Pachysolens tannophilus ATCC-32691 3 20.1

Pichia angophorae CBS-5823 6 3.0

Pichia bovis NRRL YB-4184 2 36.6

Pichia capsulate CBS- 136 4 30.7

Pichia capsulate NRRL Y- 1842 3 26.2

Pichia ciferrii NRRL Y-1031 6 3.4

Pichia guilliermondii* NRRL Y-2075 2 40.0

Pichia haplophila NRRL Y-7860 6 5.1

Pichia heimii CBS-6139 5 5.6

Pichia holstii** ATCC-13689 5 11.5

Pichia holstii CBS-2026 6 5.4

Pichia kodamae CBS-7081 5 21.9

Pichia kodamae NRRL Y-17234 4 25.8

Pichia methanolica ATCC-46071 2 26.9

Pichia methanolica CBS-6515 1 27.5

Pichia mississippiensis NRRL YB- 1294 2 30.0

Pichia naganishii ATCC-32148 2 35.5

Pichia naganishii CBS-7259 2 38.6

Pichia nakazawae var. nakazawae NRRL Y-7903 5 12.8

Pichia philogaea NRRL Y-7813 3 21.7

Pichia rabaulensis CBS-6797 1 38.7

Pichia scolyti CBS-4802 2 29.1

Pichia scolyti NRRL Y-5512 1 45.8

Pichia silvicola NRRL Y- 1679 3 20.8

Pichia stipitis ATCC-62970 6 9.0

Pichia stipitis CBS-5773 6 8.0

Pichia tannicola NRRL Y-7499 6 7.3

Pichia trehalophila CBS-5361 2 33.1

Pichia triangularis CBS-4094 3 31.4

Pseudozyma flocculosa CBS-167.88 5 6.6

Psuedozyma fusiformata CBS-6951 6 1.3

Pseudozyma rugulosa CBS-170.88 3 21.8

Rhodosporidium sphaerocarpum CBS-5939 3 27.7

Rhodotorula acuta ATCC-42713 3 32.4

Rhodotorula fragaria CBS-6254 4 16.0

Rhodotorula pustula ATCC-32034 6 6.2

Sirobasidium intermedium CBS-7805 4 15.9

Smithiozyma japonica CBS-7319 4 13.2

Sporidiobolus ruineniae CBS-5001 6 2.5

Stephanoascus smithiae CBS-5657 3 37.6

Sterigmatomyces elviae CBS-5922 3 29.3

Sympodiomycopsis paphiopedili CBS-7429 3 27.5

Tremella aurantia CBS-6965 4 15.1

Tremella cinnabarina CBS-8234 6 2.5

Tremella enchephala CBS-6968 3 20.7

Tremella foliacea CBS-8228 6 1.0

Tremella fuciformis CBS-8225 6 3.3

Tremella indecorata CBS-6976 4 15.5

Tremella nivalis CBS-8487 3 25.3

Trichosporon laibachii ATCC-90037 3 19.7

Trichosporon laibachii CBS-2495 3 16.7

Trichosporon loubieri ATCC-56048 3 24.5

Trichosporon loubieri CBS-7719 4 20.7

Trichosporon moniliiforme ATCC-22164 3 22.9

Trichosporon moniliiforme CBS-2820 3 29.1

These strains were not analyzed at 18 hours; some of them could be in group 1. **These strains were not analyzed at 144 hours; some of them could be in group 4.

Table 5. Utilization of L-arabinose by yeast strains

Group Criteria No. of strains

1 Used > 90% of available L-arabinose within 18 hours 9

2 Used all L-arabinose within 24 hours 27

3 Used all L-arabinose within 48 hours 51

4 Used all L-arabinose within 144 hours 24

5 Used > 10% of available L-arabinose in 144 hours 17

6 Did not use any or < 10% of available L-arabinose in 144 hours 37

[00122] Mutagenesis of selected strains. Our ultimate objective for these studies was not only to characterize L-arabinose utilization and uptake by various yeast species, but to identify the strains that contain efficient L-arabinose transporter(s) that could be targeted for isolation and engineering for expression in S. cerevisiae (See Examples 2-9). Since use of appropriate mutants is one of the approaches for identifying and isolating genes of interest, we examined the colony and cell morphology of the strains collected for these studies and chose only the strains that would be expected to be amenable to the application of genetic and biochemical methods. For example, we excluded strains such as Bullera penniseticola, Pichia

capsulate, Pichia kodamae, Smithiozyma japonica, Sterigmatomyces elviae, and the Myxozyma and Tremella species that formed slimy colonies and would be difficult to replica plate. We also excluded strains such as Ambrosiozyma monospora, Trichosposon laibachii, and the Psuedozyma species that tended to display significant mycelial form of growth from which it would be difficult to obtain single colony mutants. Since very limited or no genetic information is available for most of the yeast species collected for this investigation, we chose several strains, mainly from groups 1 and 2, for mutagenesis to determine if they would yield auxotophic mutants at a reasonable frequency. It is clear that a variety of mutants can be isolated from each of the selected strains with reasonable frequency (Table 6). We therefore concluded that it would be possible to isolate appropriate mutants from one or more of the organisms that were determined to harbor suitable L-arabinose transporter(s). Screening through approximately 155,000 colonies for Arxula adeninivorans, 132,000 for Debaryomyces hansenii var.fabryii, and 76,400 for Pichia guilliermondii, we also isolated mutants that were unable to grow on L-arabinose (See Fig. 1, Fig. 2, and Fig. 3). These mutants were further tested for growth on arabitol, xylose, and xylitol in an attempt to identify class of mutants expected to contain L-arabinose transport deficient mutants for use in future studies.

Table 6. EMS-induced mutagenesis of selected strains

Species Strain No of No of Mutant Types of auxotrophs colonies auxotrophs frequency obtained tested obtained (%)

A adeninivorans CBS-8244 14440 5 0 03 Ade , Arg , Met

C NRRL YB- 1299 19750 13 0 07 Ade , Arg , Lys arabinofermentans

D hansenn var CBS-2753 24400 26 0 11 Ade , Arg , His , He , fabryu Leu , Lys , Met , Thr ,

Tip , Tyr

D nepalensis NRRL Y-7108 2760 5 0 18 Ade , Arg

K marxianus CBS-712 32650 4 0 01 Ade , VaI

P guilliermondii NRRL Y-2075 6920 10 0 14 Ade , Arg , Lys , Thr ,

Tyr

P methanolica CBS-6515 27200 13 0 05 Ade , Arg , Lys , Met

[00123] Transport of L-arabinose. For our initial transport studies, l- ¹⁴ C-labeled L- arabinose from American Radiolabeled Chemicals Inc. was used as this was the only supplier from where L-(l- ¹⁴ C)arabinose was available. However, our TLC analysis of the chemical obtained from this source showed that it contained a mixture of L-arabinose and other unknown compounds and that L-arabinose constituted only a small fraction of these chemicals (Fig. 4). We did not attempt to identify the unexpected compounds present in the L-arabinose preparations. In the chromatogram of the L-(l- ¹⁴ C)arabinose sample, we have indicated the positions to which other related compounds are expected to migrate in this TLC system (Fig. 4). It should be noted that the D-(l- ¹⁴ C)xylose and D-(l- ¹⁴ C)galactose obtained from the same source are essentially pure (Fig. 4). Additionally, we used the method described by Sturgeon to establish that the compound migrating to the position expected for L-arabinose was , in fact, L-arabinose. This method uses galactose dehydrogenase to convert L-arabinose to L-arabino-lactone which is then converted to L-arabinonic acid in aqueous solution (Sturgeon, 1986). The spot expected to be L-arabinose disappears when the preparation is treated with galactose dehydrogenase while the other spots remain unaffected (Fig. 5). Subsequently, we obtained L-(l- ¹⁴ C)arabinose from custom synthesis by Moravek Biochemicals Inc. This preparation was also analyzed by the methods described above and was found to be free of contaminating compounds (data not show).

[00124] In order to select the eventual strain(s) for isolation of an L-arabinose transporter, we evaluated L-arabinose transport rates in several strains deemed suitable for further investigation (Table 7). We chose these strains based on their L-arabinose utilization efficiency (group 1-3) and suitability for genetic analysis and mutagenesis. We also tested whether L-arabinose transport in these strains is active by measuring transport in the presence of 2,4-dinitrophenol (DNP), an inhibitor of active transport. Transport of L-arabinose was inhibited by DNP in all of the strains tested indicating that these strains harbor active transport mechanisms for L-arabinose (Table 7).

Table 7 Rates of L-arabinose transport (nmole mg ^' Wn ¹ )

Group Species Source L-arabinose concentration Inhibition by (niM) 1.25 mM DNP 133 13.3 1.3 (%) ^a

1 A.adeninivorans CBS-8244 32.4±0.5 4.4±0.3 5 .2±0 .2 96

2 C.arabinofermentans NRRL YB-1299 10.8±0.0 12.6±0.4 11.5±1.9 99 ^b

2 C.blankii ATCC-18735 2.6±0.3 1.6±0.4 0.4±0.2 96

3 Dhansenii CBS-941 2.7±0.1 2.3±0.1 1.7±0.1 89

1 D.hansenii var. CBS-2753 14.2±2.5 15.2±0.7 13.3±1.5 98 fabryii

1 D.nepalensis NRRL Y-7108 5.0±2.0 3.8±0.8 3.3±0.1 99

3 K.marxianus CBS-712 4.8±0.2 5.7±0.6 1.2±0.1 88

2 K.marxianus CBS-1089 28.5±0.1 20.5±1.3 20.2±0.6 96 ^C

2 K.marxianus CBS-1555 4.4±0.3 4.5±0.9 l .O±O.O 94

2 P. bovis NRRL YB-4184 10.0±2.2 7.7±0.5 7.9±0.7 91

2 P.guilliermondii NRRL Y-2075 8.2±8.0 16.8±3.0 22.0±1.8 100 ^d

1 P.methanolica CBS-6515 14.2±1.3 10.6±0.7 9.2±0.1 99 ^b

1 P.scolyti NRRL Y-5512 4.6±0.8 4.1±0.1 2.9±0.2 94 ^a Transport inhibition assayed at 10.0 niM unless indicated. ^b Transport inhibition assayed at 1.3 niM. ^c Transport inhibition assayed at 3.0 niM. ^d Transport inhibition assayed at 0.3 niM.

[00125] Detailed transport studies with A. adeninivorans, D. hansenii var. fabryii,

Kluyveromyces marxianus, and P. guilliermondii. Based on the relatively high L-arabinose transport velocities found in our initial transport studies, we selected A. adeninivorans, D. hansenii var. fabryii, K. marxianus (CBS-1089), and P. guilliermondii for detailed L- arabinose transport studies. In the case of A. adeninivorans, both low and high affinity transport systems are present as indicated by the non-linear Eadie-Hofstee plot (Fig. 6, panel A). The low affinity transport system has a K _m of 250 mM and a V _max of 20.0 nmol/mg-min while the high affinity system has a K _m of 0.3 mM and a V _max of 6.7 nmol/mg-min. In our initial screen of transport activity and inhibition of transport in A. adeninivorans, an L- arabinose concentration of 10 mM was used (Table 7). At this low level of substrate, only the high affinity transport system would be impacted showing that this component is an active transport system.

[00126] The linear Eadie-Hofstee plots for D. hansenii var. fabryii, K. marxianus, and

P. guilliermondii indicate single component, high affinity transport systems were responsible for L-arabinose uptake (Fig. 6, panels B, C, and D respectively). These transport systems had K _m values of 0.10 mM, 0.14 mM and 0.07 mM and V _max values of 15.0 nmol/mg-min, 24.0 nmol/mg-min, and 22.5 nmol/mg-min for D. hansenii wax. fabryii, K. marxianus, and P. guilliermondii, respectively. These high capacity transport systems allowed these strains to effectively metabolize 20 g/L of L-arabinose within 18 h for A adeninivorans and D. hansenii var. fabryii and within 24 h for K. marxianus and P. guilliermondii.

DISCUSSION

[00127] There is very high degree of variability in the extent and the rate of L- arabinose utilization among various species of yeast. This example provides screening of a variety of strains for the purpose of isolating an efficient L-arabinose transporter by reasoning that strains that grew well on L-arabinose would have an efficient transport mechanism for this sugar. It was determined that of the strains may also have efficient L-arabinose transporter(s), but don't grow well on the sugar due to other defects in the L-arabinose utilization pathway. An examination of Table 4 reveals that there is significant variability in the efficiency of L-arabinose utilization among the various strains of the same species. For example, various strains of D. hansenii, which has been shown to be polyphyletic and highly variable at the genomic level (Corredor, et al., 2003; Kurtzman and Robnett, 1997), can be classified in any one of the groups 1, 3, 4, or 5. On the other hand, some genera and species showed little or no diversity. The 3 strains of C. arabinofermentans , 2 strains of C. diddensiae, 3 strains of C. succiphila, and 5 strains of K. marxianus are in similar groups (group 2 or 3). Similarly, 5 of the 6 strains of the Trichosporon sp. are in group 3 and the remaining one is in group 4. All strains of A. monospora, C. auringiensis, C. bertae, C. membranifaciens, C. shehetae, C. curvatus, F. penicillatus, P. holstii, P. naganishii, P. stipitis, T. laibachii, and T. moniliiforme were assigned to the same group as the other strains of the same species. Generally a strain from one collection had similar growth properties as the equivalent strain from a different collection. For example, A. monospora (NRRL Y1484 and CBS-2554), C. membranifaciens (NRRL Y-2089 and CBS-195), and iC. marxianus (NRRL Y-8281 and CBS-712) behaved identically from both collections. However, there were instances where the equivalent strains from different collections had somewhat different growth properties as is the case for P. kodamae (NRRL Y- 17234 and CBS-7081) and P. scolyti (NRRL Y-5512 and CBS-4802). The strains that were previously found to ferment L- arabinose grew well on L-arabinose in our screening (Dien, et al., 1996; Kurtzmann and Dien, 1998). In our study, three of these strains utilized 20 g/L of L-arabinose in 24 h (group 2) while C. auringiensis consumed all L-arabinose in 48 h (group 3). [00128] Initial uptake rates varied widely for strains in groups 1-3. No clear relationship could be established between the initial uptake rates and the time it took to completely exhaust the L-arabinose from the culture media. Strains in group 1 did not always have higher transport rates than those in groups 2 or 3. The transport of the sugar is, of course, just one (first) step in its metabolism and the velocities of other enzymes in the

pathway, the flux rates of the intermediates of the pathway, and various metabolic regulatory mechanisms contribute to the rate at which L-arabinose would be utilized by the organisms. [00129] Three strains were selected to determine if mutants unable to utilize L- arabinose for growth could be isolated. A number of such mutants were isolated from each of these strains (Fig. 1-3). Except for the mutants that do not grow on xylitol, all the mutants can be explained by a single mutation in one of the steps in the L-arabinose utilization pathway in yeasts (Chiang and Knight, 1960) (Fig. 7). This pathway has been confirmed to be functional by demonstrating that S. cerevisiae expressing genes for each of the enzymatic steps can utilize L-arabinose for growth (Richard, et al, 2003). The mutants that do not grow on xylitol may have defects at more than one step or, alternatively, there is yet an unknown pathway for xylose utilization.

[00130] The only known yeast transporter for L-arabinose, galactose permease

(Gal2p) of S. cerevisiae, is a facilitated diffusion permease and transports L-arabinose as long as the external concentration of L-arabinose is higher than the internal cellular concentration (Cirillo, 1968; Kou, et al., 1970) with a transport velocity of 0.32 nmol/mg-min at 10 mM L- arabinose (Becker and Boles, 2003). It was shown previously that Gal2p is a very high capacity L-arabinose transporter, but only at high concentrations of L-arabinose (Knoshaug et. al., L-arabinose and D-galactose transport by the galactose permease of Saccharomyces cerevisiae, submitted to Applied and Environmental Microbiology). Suprisingly, the L- arabinose transport rates in the non-conventional yeast strains were higher than that reported for Gal2p in S. cerevisiae at low concentrations of L-arabinose. In particular, the active, high affinity transport systems of K. marxianus and P. guilliermondii had transport velocities that were, respectively, 64 and 53 times greater than reported velocities for Gal2p of S. cerevisiae. [00131] Interestingly, A. adeninivorans has a combination of transporters. The combination of low and high affinity, high capacity L-arabinose transport systems endogenously present in A. adeninivorans allows for the complete assimilation of 20 g/L of L-arabinose within 18 h demonstrating that, through the combination of transport systems with different affinities for high and low concentrations of L-arabinose, a strain can be developed that can utilize L-arabinose quickly.

[00132] Two particular strains K. marxianus and P. guilliermondii of yeast were selected for further characterization and identification of single component arabinose transport.

Example 2 — Cloning of the new transporter gene KmLA Tl

[00133] A K. marxianus genomic library was constructed in our yeast vector pBFY13 which contains the yeast 2μ origin of replication, a URA3 selection cassette, and a BamHI site located between the PGKl promoter and GALlO terminator. After partial digestion of 200 μg of genomic DNA with Sau3AI restriction enzyme, fragments of 2-8 kb in length were gel- isolated and ligated into the BamHI site of pBFY013. This ligation reaction was then transformed into E. coli and plated for recovery. Plate counts produced ~3000 cfu's/10 μl of transformed cells and the plasmid DNA from 24 colonies was screened for presence of insert revealing 22 of 24 transformants had an insert ranging from 1 kb to ~8 kb giving an average insert size of 3.2 kb. The transformed cells were scraped from the plates, DNA recovered, and 5 μl was transformed into competent BF Y518 cells. The strain, BFY518, was cured of the GAL2 over-expression plasmid negating its ability to form colonies on agar plates containing L-arabinose as the sole carbon source enabling restoration of colony formation by complementation with a heterologous L-arabinose transporter. To count the number of transformed yeast cells, 10 μl of the yeast library transformation were plated onto minimal glucose media yet the colonies were so dense that only an estimate of -5000 colonies was possible. The rest of the transformation mix (~140 μl) was plated onto minimal media containing 2% L-arabinose for selection from which a small amount of background growth was noticed. The plates were then replica plated to fresh L-arabinose minimal media. The total number of cells plated for selection represented -280,000 transformants representing ~8 fold coverage of the 10.7 mb K. marxianus genome (See Dujon et al., 2004). Two colonies grew on the replica plates and the plasmid DNA was rescued and re -transformed into BFY518 allowing growth once again on L-arabinose confirming that the K. marxianus genomic insert carried on these plasmids was responsible for growth. Restriction analysis suggested both plasmids harbored the same insert of approximately 8.8 kb in size.

Example 3 — Sequence Analysis of the KmLATl gene

[00134] Sequencing results showed that both plasmids had identical inserts of 8838 kb containing two ORFs on the 5' end of the insert. Both of these ORFs showed strong homology to yeast sugar transporters. One transporter ORF was interrupted by a fragment of an unrelated ORF suggesting that recombination of fragments during ligation into the vector occurred in library construction (Fig. 10). Recombination of library fragments during ligation into the vector was shown by PCR walking experiments performed on K. marxianus genomic DNA. Walking was performed out of the transporter in a 5' direction and additional transporter sequence including the start codon was recovered rather than the additional

sequence from the unrelated ORF. The uninterrupted transporter ORF, termed KmLATl, was recovered twice more in another subsequent library screening. This ORF was 1668 bp in length and shared homology with high-affinity glucose transporters in particular, HGTl from K. lactis (Table 2) and showed a much closer association with high-affinity glucose transporters from non-conventional yeasts than the bacterial araE genes or S. cerevisiae hexose transporters (Fig. 8).

[00135] Transmembrane region prediction by the software Tmpred shows 12 transmembrane regions with a larger intercellular loop between regions 6 and 7 in KmLatl (Fig. 9) (See Hofmann et al, 1993), typical of Gal2p and other yeast sugar transporters having 10-12 transmembrane regions (See e.g., Alves-Araujo et al., 2004; Day et al., 2002; Kruckeberg et al., 1996; Pina et al. ,2004; and Weierstall et al. 1999).

Example A-KmLA Tl Expressed in S. cerevisiae Enables Growth on Arabinose

[00136] The coding sequence of KmLATl was isolated by PCR from genomic DNA of

K marxianus and cloned into a yeast 2μ plasmid under control of the PGKl promoter of S. cerevisiae. This construct was transformed into a GAL2 deleted strain of S. cerevisiae adapted to L-arabinose, BFY590. Briefly, cells are grown in appropriate selective glucose minimal media until saturation then washed and diluted to a starting ODβoo of 0.2 in minimal media supplemented with 2% L-arabinose. Cultures are incubated until exponential growth is observed then the cultures are diluted twice into the same media for continued growth to establish the final L-arabinose utilizing adapted strain which is purified on streak plates. Control plasmids carrying the yeast GAL2 gene and an empty vector were also used to transform yeast cells.

[00137] Yeast cells with a 2μ plasmid carrying the KmLATl or GAL2 gene or cells with an empty 2μ plasmid were grown with shaking in liquid minimum media containing 2% L-arabinose as the sole carbon source. The OD ₆ oo of each culture was measured and monitored by 140 hours. Growth curve results show that KmLATl is sufficient to support growth on L-arabinose when compared to cells harboring the empty vector which does not show any signs of growth (Fig. 11). This result confirms that the KmLATl gene encodes an arabinose transporter that enables yeast cells to grow on L-arabinose.

Example 5 — Comparison of the Arabinose Transport Kinetics between Gal2p and KmLatl expressed in S. cerevisiae

[00138] The transport characteristics of the KmLatl and the Gal2p transporters expressed in S. cerevisiae were compared. Both transporters were expressed in a host, BFY590, adapted for growth on L-arabinose in which the endogenous copy of GAL2 had been entirely replaced with a HIS 3 selection marker. The KmLatl transporter showed a low- affinity transporter having a Km = 230 mM and a Vmax = 55 nmol/mg-min (Fig. 12A). This is in contrast to the high-affinity active transport activity induced in the wild type K. marxianus when grown on 2% L-arabinose (Fig. 12B). These results suggest there are at least 2 transporters in K. marxianus that may transport L-arabinose but only the high-affinity activity is induced in the wild type when grown on 2% L-arabinose. Inhibition experiments showed that KmLatl expressed in S. cerevisiae is not significantly inhibited by protonophores such as NaN3, DNP, and CCP. Neither is KmLatl inhibited by H+-adenosine triphosphatase (ATPase) inhibitors such as DESB and DCCD (Table 8). This is in contrast to the transport activity in wild type K. marxianus, suggesting that KmLatl is a facilitated diffusion permease similar to the Gal2 permease. Competition experiments showed that KmLatl is out-competed by glucose, galactose, xylose, and maltose when expressed in S. cerevisiae (Table 8).

Table 8. Effect of Inhibitors or Competing Sugars on the Rate of

L-Arabinose Transport in L-Arabinose-Grown S. cerevisiae

Expressing GAL2 or KmLATl

Inhibitor or Concentration(mM) Relative L-arabinose

Competing Sugar transport (%)

Gal2 KmLatl

None NA 100 ^a 100 ^c

NaN ₃ 10 66 11

CCCP 5 46 61

DCCD 5 69 55

DNP 5 72 75

DESB 5 81 100

None NA 100 ^b 100 ^d

Glucose 900 10 17

Galactose 900 3 23

Xylose 900 25 25

Maltose 450 ND 38

^a Uptake rate was 66.0 nmol mg ^"1 min ^"1 determined with 118 mM labeled L- arabinose. ^b Uptake rate was 18.9 nmol mg ^"1 min ^"1 determined with 30 mM labeled L- arabinose. ^c Uptake rate was 7.7 nmol mg ^"1 min ^"1 determined with 118 mM labeled L- arabinose.

^d Uptake rate was 3.6 nmol mg ^"1 min ¹ determined with 30 niM labeled L- arabinose.

ND, Not Done.

[00139] Transport kinetics of S. cerevisiae BFY597 over-expressing the Gal2 permease grown on 2% L-arabinose showed a K _m of 550 mM and a V _max of 425 nmol/mg-min for L-arabinose transport (Fig. 12A). Inhibition assays showed a reduction but not a complete inhibition of transport suggestive of facilitated diffusion transport as previously reported (Kuo et al. 1970, see also Table 4). Competition studies showed that glucose, galactose, and xylose significantly reduced L-arabinose transport indicating that these sugars are preferentially transported over L-arabinose (Table 8). The kinetics of galactose transport were also measured in this strain and indicate that Gal2p has a K _m of 25 mMand a V _max of 76 nmol/mg-min for galactose transport, demonstrating a higher affinity for galactose that would out-compete L-arabinose for transport.

Example 6 — Cloning of the New Transporter Gene PgLAT2

[00140] Pichia guilliermondii cells were grown in minimal media supplemented with

2% L-arabinose, galactose, or xylose. Cells were collected in mid-growth and washed twice in water before suspension in water at about 30 mg/ml. RNA was extracted from the cells using the acid phenol method (Ausubel, et al., Short Protocols in Molecular Biology, John Wiley and Sons, 1999). Briefly, approximately 15 mL of fresh culture was added to -25 mL of crushed ice and centrifuged at 4° C for 5 min at 3840 x g. Cells were washed twice with cold DEPC-treated water, and the pellets were frozen at -80° C. After the pellets were resuspended in 400 ul TES (10 mM Tris HCl, pH 7.5, 5 mM EDTA, 0.5% SDS), 400 ul of acid phenol was added. The samples were vortexed vigorously for 10 sec, followed by incubation for 30-60 min at 65° C with occasional vortexing. The tubes with the samples were then chilled on ice and spun for 5 min at 4° C. The aqueous phase was removed and re- extracted with chloroform. The aqueous phase was then ethanol precipitated using 0.1 volume of 3 M sodium acetate (pH 5.3) and two volumes of 100% ethanol. The pellet was washed using 80% ethanol, dried, and resuspended in 50 ul DEPC H ₂ O. Total RNA concentration was quantitated by measuring the OD ₂ 6o and visualized on agarose gels. [00141] RNA purification, synthesis of cDNA, and differential display were performed at GenHunter Corporation according to standard techniques. DNA Bands showing higher levels of expression from arabinose-grown cells relative to xylose- or galactose-grown cells were reamplified using the differential display amplification primers. Direct sequencing was

performed on the PCR products using the GenHunter arbitrary primers. In cases that did not yield clean sequence, the amplification products were cloned in the TOPO-TA vector pCR2.1 (Invitrogen) and individual clones were sequenced. Sequences were then compared to the databases using BLASTX analysis and those that showed similarity to known transporters or transporter-like proteins were examined further. One of these sequences led to the identification of a novel transporter gene, PgLAT2 from Pichia guilliermondii. [00142] The full-length PgLAT2 gene was isolated by genome walking in P. guilliermondii. PCR-based walking was done in both the 5' and 3' directions from the sequence isolated by differential display. The entire gene was then isolated from genomic DNA using PCR primers based on the flanking DNA sequences. PgLAT2 gene has an ORF of 1617 nucleotides encoding a protein with a predicted length of 539 amino acids (Fig. 13). Sequence similarity was observed between PgLAT2 and other sugar transporter genes, including high affinity glucose transporters from Candida albicans and Kluyveromyces lactis. Similar to KmLatl, the predicted PgLat2 polypeptide showed 12 transmembrane regions with a larger intercellular loop between regions 6 and 7, typical of yeast sugar transporters.

Example 7 — Characteristics of Sugar Transport by Pichia guilliermondii [00143] The induction of L-arabinose transport in wild type P. guilliermondii was examined. Wildtype Pichia guilliermondii cells were grown in minimal media supplemented with 2% L-arabinose, galactose, or xylose while BFY605 cells were grown in the same media supplemented with 0.2% L-arabinose. Cells were collected in mid-growth and washed twice in water before suspension in water at about 30 mg/ml. Uptake of L-(I- ¹⁴ C)arabinose (54 mCi/mmol, Moravek Biochemicals Inc.), D-(l- ¹⁴ C)galactose (57 mCi/mmol, Amersham Biosciences), or D-(l- ¹⁴ C)xylose (53 mCi/mmol, Moravek Biochemicals Inc.) was measured as previously described (Stambuk, Franden et al. 2003). Assays were performed in 5, 10, or 30 second periods to maintain initial rates. Appropriate experiments ensured uptake was linear for at least 1 minute. Transport activity was described as nmoles of labeled sugar transported per mg cell dry weight per minute. Inhibition and competition assays were performed as previously described (Stambuk, Franden et al. 2003).

[00144] Cells grown on L-arabinose were able to transport L-arabinose whereas cells grown on galactose or xylose were not able to transport L-arabinose. Additionally, xylose transport was about double in cells grown in L-arabinose media compared to cells grown in xylose media. Galactose was transported at the same rate independent of growth substrate (Fig. 14). Transport competition between L-arabinose and xylose was also examined.

Uptake of labeled L-arabinose was reduced by 96% when 10Ox un-labeled xylose was included in the transport assay whereas uptake of labeled xylose was only reduced by 16% when 10Ox un-labeled L-arabinose was included in the assay (Fig. 15). These data suggest that in P. guilliermondii, growth on L-arabinose induces expression of a specific transport system capable of transporting L-arabinose and xylose. Additionally, this system preferentially transports xylose at the expense of L-arabinose if both sugars are present and has a higher transport velocity for xylose than the transport system induced when grown on xylose. By contrast, transport activity for L-arabinose is not induced when grown on xylose.

Example 8 — Arabinose Transport Kinetics of PgLat2 expressed in S. cerevisiae

[00145] The L-arabinose transport characteristics of the PgLat2 transporter expressed from a 2μ plasmid under control of the PGKl promoter of S. cerevisiae in S. cerevisiae grown on 0.2% L-arabinose medium showed the same L-arabinose transport characteristics as wildtype P. guilliermondii (Fig. 16). The PgLat2 transporter when expressed in S. cerevisiae has a K _m = 0.07 mM and V _max = 18 nmol/mg-min. Inhibition experiments showed significant inhibition of transport by protonophores (NaN3, DNP, and CCP) and H+- adenosine triphosphatase (ATPase) inhibitors (DESB and DCCD) similar to the inhibition observed in wildtype P. guilliermondii (Table 9). Competition experiments showed that L- arabinose uptake by the PgLat2 transporter was inhibited by glucose, galactose, xylose and to a lesser extent by maltose (Table 9).

Table 9

Effect of Inhibitors or Competing Sugars on the Rate of L-Arabinose Transport in L-Arabinose-Grown P. guilliermondii Y-2075 and S. cerevisiae BFY605

Inhibitor Concentration Relative L-arabinose transport or (mM)

Competing P. guilliermondii S. cerevisiae (PgLaU

Sugar transporter)

None ³ - 100 100

NaN ₃ 10 1 16

DNP 5 0 4

CCCP 5 0 2

DCCD 5 22 36

DESB 5 8 1

None ^b - 100 100

Glucose 120 ND 17

Galactose 120 ND 20

Xylose 120 4 0

Maltose 120 ND 30

^a Rate of L-arabinose transport was 11.2 nmol mg ^"1 min ^"1 for P. guilliermondii and 10.4 nmol mg ^"1 min ^"1 for S. cerevisiae (PgLat2 transporter) determined with 0.33 mM labeled L- arabinose.

Rate of L-arabinose transport was 14.2 nmol mg ^"1 min ^"1 for P. guilliermondii and 14.4 nmol mg ^"1 min ^"1 for S. cerevisiae (PgLat2 transporter) determined with 1.2 mM labeled L- arabinose.

[00146] The transport activities, inhibition profiles, and competition rates with respect to xylose of wildtype P. guilliermondii and of the PgLat2 transporter expressed in S. cerevisiae are very similar, suggesting that P. guilliermondii has a single, high affinity, active transporter charged with uptake of L-arabinose. There are no L-arabinose transport activities that are unaccounted for which suggests the presence of a single L-arabinose transporter in P. guilliermondii.

Example 9 — Synergistic Effect on Growth Rate and Sugar Utilization by S. cerevisiae Expressing Gal2p and the New Transporter Protein-PgLat2 [00147] To determine the complementary effects on arabinose transport by the transporters, namely, Gal2p and PgLat2, yeast strains were constructed with appropriate selection markers to allow different pathway and transporter combinations to be expressed. Transporter combinations were generated by introducing a transporter expression plasmid for PgLAT2 (or an empty vector) into S. cerevisiae strain BFY607 expressing the bacterial genes

araA, araB and araD (See e.g., Becker and Boles, for examples of yeast strain expressing these bacterial proteins for arabinose metabolism). The resulting strains, expressed Gal2p due to the gal80- genotype that de -represses GAL2 expression. Strains BFY609 and BFY612 containing a control vector or a PgLat2 expression vector, respectively, were able to grow on 2% or 0.2% L-arabinose after extensive lag times (A process termed "adaptation."). A relatively low concentration of L-arabinose (0.2%) was used in this experiment as strain differences are more pronounced at this concentration. Once "adapted" to growth on 0.2% L- arabinose, the strains were able to grow more quickly and growth curves for the two transporter combination were generated as shown in Fig. 17 (Also see Table 3). A significant lag time was observed due to their inoculation from stationary cultures. However, once growth initiated, the growth rate was relatively rapid. The doubling time for each culture in the exponential phase of the curve is shown in Table 3. The doubling time for the PgLat2 and Gal2p co-expressing cells was markedly shorter than in the cells expressing only Gal2p (15 hours vs. 19 hours). A second observation relates to the overall extent of growth. The PgLat2 expressing strain appeared to grow to a higher overall optical density at saturation, suggesting that this strain was able to utilize the carbon source (L-arabinose) in the medium more completely (Fig. 17).

Example 10 — Co-expression of Gal2p with PgLat2 Enables more Complete Utilization of Arabinose by Recombinant S. cerevisiae

[00148] Doubling times for the cultures described above in Example 9 were measured in early exponential phase for each culture. Doubling time was measured by the period of time taken for the number of cells to double in a given cell culture (See generally, Guthrie and Fink, 1991). The concentration of remaining L-arabinose at the 276 hour time point was determined by HPLC (for saturated cultures only). The concentration of L-arabinose in the starting media was about 1.89 g/L and the concentration of L-arabinose in media without L- arabinose had an undetectable level (<0.1 g/L). As shown in Table 3, significantly less residual L-arabinose remained in the culture of cells expressing both Gal2p and PgLat2 than in the culture of cells expressing Gal2p alone.

[00149] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.

[00150] This specification contains numerous citations to references such as patents, patent applications, and scientific publications. Each is hereby incorporated by reference for all purposes.

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