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1. (WO2015069796) METHODS FOR ENGINEERING SUGAR TRANSPORTER PREFERENCES
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METHODS FOR ENGINEERING SUGAR TRANSPORTER

PREFERENCES

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/900,1 15, filed November 5, 2013, which is hereby incorporated by reference in its entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under grant number CBET- 1067506 awarded by the National Science Foundation. The Government has certain rights in this invention.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER

PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

[0003] The Sequence Listing written in file 93331-920858_ST25.TXT, created on November 3, 2014, 12,049 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0004] The quest for an optimal xylose pathway in yeast is of utmost importance along the way to realizing the potential of lignocellulosic biomass conversion into fuels and chemicals. An often overlooked aspect of this catabolic pathway is the molecular transport of this sugar.

Molecular transporter proteins facilitate monosaccharide uptake and serve as the first step in catabolic metabolism. In this capacity, the preferences, regulation, and kinetics of these transporters ultimately dictate total carbon flux. Optimization of intracellular catabolic pathways only increases the degree to which transport exerts control over metabolic flux. Thus, monosaccharide transport profiles and rates are important design criteria and a driving force to enable metabolic engineering advances. Among possible host organisms, Saccharomyces cerevisiae is an emerging industrial organism. However, S. cerevisiae lacks an endogenous xylose catabolic pathway and thus is unable to natively utilize the second most abundant sugar in lignocellulosic biomass, xylose. Decades of research have been focused on improving xylose

catabolic pathways in recombinant 5*. cerevisiae, but little effort has been focused on the first committed step of the process— xylose transport, an outstanding limitation in the efficient conversion of lignocellulosic sugars. There is a need in the art for efficient transport systems for xylose in yeast. Provided herein are solutions to these and other problems in the art.

BRIEF SUMMARY OF THE INVENTION

[0005] Accordingly, provided herein, inter alia, are compositions and methods useful for transporting xylose, arabinose, galactose and other monosaccharides and polysaccharides into a yeast cell.

[0006] In a first aspect is a recombinant xylose transporter protein including a transporter motif sequence corresponding to amino acid residue positions 36, 37, 38, 39, 40, and 41 of

Candida intermedia GXSl protein. The transporter motif sequence is -G-G/F-X1-X2-X3-G-. X1 is D, C, G, H, I, L, or F. X2 is A, D, C, E, G, H, or I. X3 is N, C, Q, F, G, L, M, S, T, or P. The transporter motif sequence is not -G-G-L-I-F-G- or -G-G-F-I-F-G-.

[0007] In another aspect is a recombinant galactose-arabinose transporter protein including a transporter motif sequence corresponding to amino acid residue positions 36, 37, 38, 39, 40, and 41 of Candida intermedia GXSl protein. The transporter motif sequence is -G-G/F-X4-X5-X6-G-. X4 is D, C, F, G, H, L, R, T, or P. X5 is A, C, E, F, H, K, S, P, or V. X6 is R, D, E, F, H, I, M, T, or Y. The sequence is not -G-G-L-V-Y-G-, or -G-G-F-V-F-G-.

[0008] Also provided herein are yeast cells that include a recombinant hexose or pentose transporter protein described herein. In one aspect the yeast cell includes a recombinant xylose transporter protein described herein. In another aspect the yeast cell includes a recombinant galactose-arabinose transporter described herein.

[0009] Provided herein are nucleic acid sequences that encode a recombinant hexose or pentose transporter protein described herein. In one aspect the nucleic acid encodes a

recombinant xylose transporter protein described herein. In another aspect the nucleic acid encodes a recombinant galactose-arabinose transporter protein described herein.

[0010] Further provided herein are methods of transporting a hexose or pentose into a yeast cell using the recombinant transporter proteins described herein. In one aspect is a method of transporting xylose into a yeast cell by contacting a yeast cell having a recombinant xylose transporter protein described herein with a xylose compound described herein. The xylose transporter protein is allowed to transport the xylose compound into the yeast cell. In another aspect is a method of transporting galactose or arabinose into a yeast cell by contacting a yeast cell having a recombinant galactose-arabinose transporter protein described herein with a galactose compound or an arabinose compound described herein. The recombinant galactose-arabinose transporter protein is allowed to transport the galactose compound or the arabinose compound into the yeast cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 - Sequence categorization and phenotypic classification of native and heterologous transporters. A) The distribution of phenotypic classes for 46 cloned wild type major facilitator superfamily transporters. B) The distribution of each sequence category present in each phenotypic class. Transporters containing the conserved motif are enriched in the phenotypic classes that confer growth on xylose. C) Weblogos of the phenotypic classes illustrate enrichment of the G-G/F-XXXG motif in TMS1. Abbreviations: μα\\ = 0: no growth the five carbon sources tested. μχ = 0: growth on hexoses but not xylose. μχ < μο: growth on xylose is less than that on glucose, μχ > μο: growth on xylose is greater than that on glucose.

[0012] Figure 2 - Classification tree of fractional change in carbon source growth profile. This figure depicts hypothetical fractional change data in order to demonstrate how these phenotypes were classified. Little fractional change across all sugars indicates that the substitution does not control efficiency or selectivity in this background. Amplification or attenuation of growth rates across all carbon sources indicates an efficiency substitution. Amplification of growth on one sugar, ideally xylose, and attenuation of all others indicates a selectivity substitution.

[0013] Figure 3 - Fractional change of saturation mutagenesis libraries of C. intermedia GXS1. A) Fractional change in growth by substitutions at position 38. B) Fractional change in growth by substitutions at position 39. C) Fractional change in growth by substitutions at position 40. The solid line is the confidence line for no growth based on the negative control sample.

[0014] Figure 4 - Growth characterization of C. intermedia gxsl triple mutants. A) Fractional change from wild type for the two triple mutants and an empty vector control. B) Average growth curves on xylose based on optical density at 600 nm. C) Average growth curves on glucose based on optical density at 600 nm.

[0015] Figure 5 - Further characterization of C. intermedia gxsl Phe38 He39 Met40 triple mutant. A) Glucose uptake at high cell density for S. cerevisiae EX.12 expressing wild type,

Phe He Met , and empty vector. B) Xylose uptake at high cell density for 5*. cerevisiae EX.12 expressing wild type, Phe38 He39 Met40, and empty vector. C) Inhibition of growth rate on xylose with increasing glucose concentration. D) Vmax of both the wild type and the mutant. E) KM of both the wild type and triple mutant. Error is based on standard deviation of biological replicates.

[0016] Figure 6 - Growth characterization of S. stipitis RGT2 and mutants. A) Fractional change from wild type for the two single mutants and an empty vector control. B) Average growth curves on xylose based on optical density at 600 nm. C) Average growth curves on glucose based on optical density at 600 nm.

[0017] Figure 7 - Growth characterization of S. cerevisiae HXT7 and mutants. A) Fractional change from wild type for the mutants and an empty vector control. B) Average growth curves on xylose based on optical density at 600 nm. C) Average growth curves on glucose based on optical density at 600 nm.

[0018] Figure 8 - Maximum exponential growth rates for all cloned native and heterologous transporters. Bar chart of growth rate (μ) calculated from growth curves of S. cerevisiae EX.12 measured on a Bioscreen C. Carbon source profiling on five different sugars allows better functional classification than measuring only glucose and xylose. Error is standard deviation of biological triplicates. A) Transporters cloned in the initial study measured for the first time in 5*. cerevisiae EX.12. B) Novel transporters identified and characterized. Abbreviations: Empty -empty vector control strain. A.t. - Arabidopsis thaliana. C.i. - Candida intermedia. C.n. -Cryptococcos neoformans. D.h. - Debaryomyces hansenii. S.c. - Saccharomyces cerevisiae. S.s. - Scheffersomyces stipitis. Y.l. - Yarrowia lipolytica.

[0019] Figure 9 - High cell density cofermentation in 5*. cerevisiae EX.12. Cells were inoculated at OD 20 in a mixture of 10 g/L glucose and 10 g/L xylose. Optical density, glucose, xylose, and ethanol concentration was measured over the length of the fermentation. Note that the triple mutant does not consume either xylose or glucose, nor is an appreciable amount of ethanol produced in this multiple knockout strain. A) Optical density over time. B) Glucose concentration in the media over time. C) Xylose concentration in the media over time. D)

Ethanol concentration in the media over time.

[0020] Figure 10 - High cell density cofermentation in S. cerevisiae YSX3. Cells were inoculated at OD 20 in a mixture of 10 g/L glucose and 10 g/L xylose. Optical density, glucose, xylose, and ethanol concentration was measured over the length of the fermentation. Note that

the triple mutant does not appreciably alter the fermentation dynamics in a strain that is expressing the full suite of transporters. A) Optical density over time B) Glucose concentration in the media over time. C) Xylose concentration in the media over time. D) Ethanol

concentration in the media over time.

[0021] Figure 11 - Growth curves of transporters of interest. Optical density measurements from the Bioscreen C were plotted over time. Each line represents the growth curve for 5*.

cerevisiae EX.12 expressing a transporter on a particular carbon source. A) D.h. 2D01474. B) S.s. RGT2. C) D.h. 2E01166. D) D.h. 2B05060. E) S.c. STL1. F) S.s. AUT1.

[0022] Figure 12 - Phylogenetic tree and growth rate. Phylogram constructed in TreeView of a ClustalW multiple sequence alignment with the full amino acid sequences of all transporters. To the right of the phylogram is plotted the exponential growth rate of S. cerevisiae EX.12 conferred by transporter expression. A blue line and a green line are placed across the chart to mark the upper limit of no growth for glucose and xylose, respectively. Note the most robust glucose growth phenotypes are clustered in the HXT family and related transporters. Some of the more desirable growth phenotypes for xylose growth are clustered in the transporters related to C.i. GXS1 and S.s. XUT3.

[0023] Figure 13 - Relatedness based on G-G/F-XXXG motif and growth rate data.

Phylogram constructed in TreeView of a ClustalW multiple sequence alignment of the G-G/F-XXG motif of each transporter. To the right of the phylogram is plotted the exponential growth rate of S. cerevisiae EX.12 conferred by transporter expression. Two lines are placed across the chart to mark the upper limit of no growth for glucose and xylose. Arranging the transporters in this fashion remarkably clusters conferred phenotype better than basing the alignment on the whole amino acid sequence. This is further evidence of the influence the G-G/F-XXG motif has over monosaccharide uptake.

[0024] Figure 14- Carbon source profile comparison. A) C.i. GXS1 and mutants. B) S.s. RGT2 and mutants. C) S.c. HXT7 and mutants. Note that these values are maximum exponential growth rates, and therefore may produce different comparisons than the late-stage linear exponential portions of the growth curves.

[0025] Figure 15 - Growth characterization of C. intermedia gxsl rationally designed triple mutants. Fractional change from wild type is calcualted on a variety of carbon sources for five mutants with differing transporter motif sequences (e.g. FLS, FIS, FIM, RPT, TPT, *VP which

contains a stop codon in the motif) compared to the negative control with no transporter motif sequence. The r 38p39r 40 mutant shows a distinct preference toward galactose and away from the other sugars tested.

[0026] Figure 16 - Growth curves of rational gxsl mutants by mutation. Growth curves are presented for the mutants described in Figure 15 on glucose, xylose, galactose, fructose, and mannose. Data is presented in graphs separated by mutant.

[0027] Figure 17 - Growth curves of rational gxsl mutants by mutation. Growth curves are presented for the mutants described in Figure 15 on glucose, xylose, galactose, fructose, and mannose. Data is presented in graphs separated by carbon source.

[0028] Figure 18 - Rewiring xut3 transporter proteins through the equivalent of the 297 residue from C. intermedia GXSl. A) Identification of previously identified mutations in the xut3 mutant transporter. B) Saturation mutagenesis was performed on the equivalent of the 297 residue from C. intermedia GXSl. Fractional change on growth of various carbon sources was measured and the results illustrated that this residue can control sugar transporter preference.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are well known and commonly used in the art for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document.

[0030] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single

and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA. Nucleic acid as used herein also refers nucleic acids that have the same basic chemical structure as a naturally occurring nucleic acids. Such analogues have modified sugars and/or modified ring substituents, but retain the same basic chemical structure as the naturally occurring nucleic acid. A nucleic acid mimetic refers to chemical compounds that have a structure that is different the general chemical structure of a nucleic acid, but that functions in a manner similar to a naturally occurring nucleic acid. Examples of such analogues include, without limitation,

phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-0-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0031] "Synthetic mRNA" as used herein refers to any mRNA derived through non-natural means such as standard oligonucleotide synthesis techniques or cloning techniques. Such mRNA may also include non-proteinogenic derivatives of naturally occurring nucleotides. Additionally, "synthetic mRNA" herein also includes mRNA that has been expressed through recombinant techniques or exogenously, using any expression vehicle, including but not limited to prokaryotic cells, eukaryotic cell lines, and viral methods. "Synthetic mRNA" includes such mRNA that has been purified or otherwise obtained from an expression vehicle or system.

[0032] The words "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.

[0033] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are near each other.

[0034] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one

or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0035] The term "recombinant" when used with reference to, for example, a cell, nucleic acid, or protein, indicates that the cell, nucleic acid, or protein, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express genes otherwise modified from those found in the native form of a cell (e.g. genes encoding a mutation in a native or non-native transporter protein, such as a transporter motif sequence described herein). For example, a recombinant protein may be a protein that is expressed by a cell or organism that has been modified by the introduction of a heterologous nucleic acid (e.g.

encoding the recombinant protein).

[0036] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0037] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0038] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.

[0039] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0040] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),

Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0041] A "yeast cell" as used herein, refers to a eukaryotic unicellular microorganism carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. Yeast cells may carry out fermentation of sugars described herein. Fermentation may convert the sugar to a biofuel or biochemical as set forth herein. Yeast cells referenced herein include, for example, those species listed in Figure 19 or Figure 20. Yeast cells referenced herein include, for example, the following species: Kluyveromyces lactis, Torulaspora delbrueckii, Zygosaccharomyces rouxii, Saccharomyces cerevisiae, Yarrowia Upolytica, Candida intermedia, Cryptococcos neoformans, Debaryomyces hansenii, Phaffia rhodozyma, or Scheffersomyces stipitis.

[0042] The term "biofuel" as used herein refers to a convenient energy containing substance produced from living organisms (e.g. biomass conversion to a fuel). Thus, biofuels may be produced through, for example, fermentation of carbohydrates (e.g. sugars) found in biomass (e.g. lignocellulosic biomass). Biofuels may be solid, liquid, or gas forms. Biofuels include, for example, ethanol, biodiesel, vegetable oil, ether (oxygenated fuels), or gas (e.g. methane).

[0043] The term "biochemical" as used herein refers to chemicals produced by living organisms. Biochemicals herein include alcohols (e.g. butanol, isobutanol, 2,3-butanediol, propanol); sugars (e.g. erythritol, mannitol, riboflavin); carotenoids (e.g. β-carotene, lycopene, astaxanthin); fatty acids (e.g. ricinoleic acid, linolenic acid, tetracetyl phytosphingosine); amino acids (e.g. valine, lysine, threonine); aromatics (e.g. indigo, vanillin, sytrene, p-hydroxystyrene); flavonoids (e.g. naringenin, genistein, kaempferol, quercetin, chrysin, apigenin, luteolin,);

stillbenoids (e.g. resveratrol); terpenoids (e.g. β-amyrin, taxadiene, miltiradiene, paclitaxel, artemisinin, bisabolane); polyketides (e.g. aureothin, spectinabilin, lovastatin, geodin); or organic acids (e.g. citric acid, succinic acid, malic acid, lactic acid, polylactic acid, adipic acid, glucaric acid) produced by living organisms (e.g. a yeast cell). See e.g. Curran K.A., Alper H.S., Metabolic Engineering 14:289-297 (2012).

[0044] A "transporter motif sequence" as used herein refers to an amino acid sequence that, when present in a protein (e.g. a sugar transporter protein such as a MFS transporter protein), increases the ability of the protein to transport a sugar or sugar-containing compound into a yeast cell . The transporter motif sequence may impart a hexose sugar transport preference or pentose sugar transport preference to the protein. Thus, for example, the transporter motif sequence may impart preference to hexose sugars to a transporter protein, thereby allowing the transporter protein to preferentially transport hexoses into a yeast cell. The transporter motif sequence may impart preference to a single hexose (e.g. galactose). The transporter motif sequence may impart preference to more than one hexose sugar (galactose and mannose). The transporter motif sequence may impart preference to pentose sugars to a transporter protein, thereby allowing the transporter protein to preferentially transport pentose into a yeast cell. The transporter motif sequence may impart preference to a single pentose (e.g. xylose). The transporter motif sequence may impart preference to more than one pentose sugar (e.g. xylose and arabinose). The

transporter motif sequence may impart preference for at least two sugars (e.g. galactose and arabinose).

[0045] The transporter motif sequence described herein corresponds to residues corresponding to positions 36-41 of the Candida intermedia GXS1 protein ("GXS1 motif sequence"). One skilled in the art will immediately recognize the identity and location of residues corresponding to positions 36-41 of the Candida intermedia GXS1 protein in other transporter proteins with different numbering systems. For example, by performing a simple sequence alignment with Candida intermedia GXS1 protein the identity and location of residues corresponding to positions 36-41 of the Candida intermedia GXS1 protein are identified in other yeast transport proteins as illustrated in Figures 19 and 20. Insertion (e.g. substitution) of a transporter motif sequence into a yeast transport protein may thereby be performed resulting in a functional yeast transporter protein with an altered sugar transport preference (e.g. changing a preference for hexoses to a preference for pentoses). For example, amino acid residue positions 75-81 of S. cerevisiae HXT7 protein correspond to amino acid residue positions 36-41 of the Candida intermedia GXS1 protein. See e.g. Example 2 and SEQ ID NO: 1.

SEQ ID O: l

1 MGLEDNRMVKRFVNVGEKKAGSTAMAI IVGLFAASGGVLFGYDTG ISGVMTMDYVLARY 60

61 PSNKHSFTADESSLIVSILSVGTFFGALCAPFLNDTLGRRWCLILSALIVFNIGAILQVI 120

121 STAIPLLCAGRVIAGFGVGLISATIPLYQSETAPKWIRGAIVSCYQWAITIGLFLASCVN 180 181 KGTEHMTNSGSYRIPLAIQCLWGLILGIGMIFLPETPRFWISKGNQEKAAESLARLRKLP 240

241 IDHPDSLEELRDITAAYEFETVYGKSSWSQVFSHKNHQLKRLFTGVAIQAFQQLTGVNFI 300

301 FYYGTTFFKRAGVNGFTISLATNIVNVGSTIPGILLMEVLGRRNMLMGGATGMSLSQLIV 360 361 AIVGVATSENNKSSQSVLVAFSCIFIAFFAATWGPCAWVVVGELFPLRTRAKSVSLCTAS 420 421 NWLWNWGIAYATPYMVDEDKGNLGSNVFFIWGGFNLACVFFAWYFIYETKGLSLEQVDEL 480 481 YEHVSKAWKSKGFVPSKHSFREQVDQQMDSKTEAIMSEEASV 522

[0046] A "transporter protein" as used herein refers to a transmembrane protein which transports sugars (e.g. hexoses and pentoses) into a yeast cell. The transporter protein may be a yeast transporter protein. The transporter protein may be a transporter protein belonging to the major faciliator superfamily ("MFS") transporter proteins. A transporter protein may transport a hexose (e.g. galactose) into a yeast cell. A transporter protein may transport a pentose (e.g. xylose or arabinose) into a yeast cell. A transporter protein may be engineered, using the transporter motif sequences described herein, to alter its sugar preference (e.g. a transporter protein having a preference to transport a hexose compound may be converted to a transporter protein having a preference to transport a pentose compound). A transporter protein may be

characterized as a transporter protein derived from a particular organism. Where a transporter protein is derived from a particular organism, the endogenous sequence of the transporter protein may be maintained and residues corresponding to positions 36-41 of the Candida intermedia GXS1 protein may be replaced with a transporter motif sequence. For example, a C. intermedia gxsl transporter protein is a gxsl transporter protein, a homolog thereof, or a functional fragment thereof, found in C. intermedia SEQ ID NO: l . Amino acids 75-81 of S. cerevisiae hxtl transporter protein may be replace with a transporter motif sequence thereby forming a transporter protein with desired sugar transport characteristics described herein. The transporter protein may be a protein, functional fragment, or homolog thereof, identified by the following NCBI gene ID numbers: 836043, 831564, AJ937350.1, AJ875406.1, 2901237, 2913528, 8998057, 8999011, 50419288, 948529, 4839826, 4852047, 4851844, 4840896, 4840252, 4841106, 4851701, 2907283, 2906708, 2908504, 2909312, 2909701, 4935064, 851943, 856640, 856640, 851946, 856494, 8998297, 2902950, 2902912, 853207, 852149, 855023, 853216, 853236, 850536, 855398, 4836720, 4836632, 4840859, 2913215, 2902914, 2910370, 4838168, 2901237.

[0047] A "xylose compound" is xylose or a xylose-containing compound including at least one xylose moiety. Thus as used herein, the term xylose compound represents a single xylose, a chain including one or more xylose moieties, or a xylose moiety covalently or non-covalently bound to another chemical moiety (e.g. another sugar forming a xylose containing

polysaccharide or xylose bound to lignin). An "arabinose compound" is arabinose or an arabinose-containing compound including at least one arabinose moiety. Thus as used herein, the term arabinose compound represents a single arabinose, a chain including one or more arabinose moieties, or an arabinose moiety covalently or non-covalently bound to another chemical moiety (e.g. another sugar forming a arabinose containing polysaccharide or arabinose bound to lignin). A "galactose compound" is galactose or a galactose-containing compound including at least one galactose moiety. Thus as used herein, the term galactose compound represents a single galactose, a chain including one or more galactose moieties, or a galactose moiety covalently or non-covalently bound to another chemical moiety (e.g. another sugar forming a galactose containing polysaccharide or bound to lignin).

[0048] Polysaccharides herein include hexose-only polysaccharides, pentose-only

polysaccharides, and hexose-pentose mixture polysaccharides. The xylose compound, the arabinose compound, or the galactose compound may be derived from or form part of a

lignocellulosic biomass (e.g. plant dry matter that may used in as a source for pentose compounds or hexose compounds and for production of biofuels or biochemicals), hemicelluose, or other natural or synthetic sources for xylose, arabinose, or galactose. "Derived from" refers to extraction, removal, purification, or otherwise freeing a xylose compound, arabinose compound, or galactose compound from a source (e.g. lignocellulosic biomass) by either chemical processes (e.g. acid hydrolysis, ammonium explosion, or ionic liquids extraction) or through natural biological processes by organisms capable of using such sources for energy.

[0049] A "pentose compound" or "pentose" is a monosaccharide-containing compound having 5 carbon atoms. Pentose compounds include aldopentoses (e.g. pentose compounds having an aldehyde moiety at carbon 1) and ketopentoses (e.g. pentose compounds having a ketone moiety at carbon 2 or carbon 3). Pentose compounds include, for example, D/L-arabinose, D/L-lyxose, D/L-ribose, D/L-xylose, D/L-ribulose, and D/L-xylulose. The term "monosaccharide-containing" refers to a compound that includes at least one monosaccharide.

[0050] A "hexose compound" "or "hexose" is a monosaccharide-containing compound having 6 carbon atoms. Hexose compounds include aldohexoses (e.g. hexose compounds having an aldehyde moiety at carbon 1) and ketohexoses (e.g. hexose compounds having a ketone moiety at carbon 2). Hexose compounds include, for example, D/L-allose, D/L-altrose, D/L-glucose, D/L-mannose, D/L-gluose, D/L-idose, D/L-galactose, and D/L-talose.

[0051] The word "expression" or "expressed" as used herein in reference to a DNA nucleic acid sequence (e.g. a gene) means the transcriptional and/or translational product of that sequence. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et ah, 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88). The level of expression of a DNA molecule may also be determined by the activity of the protein.

[0052] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. A "protein gene product" is a protein expressed from a particular gene.

[0053] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including

biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product or interaction can be produced directly between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0054] The term "contacting" may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound described herein (e.g. xylose compound, arabinose compound, or galactose compound) and a protein or enzyme described herein.

Contacting may include allowing the compound described herein to interact with a protein or enzyme that is involved in transporting hexose compounds or pentose compounds into a yeast cell.

[0055] Provided herein are recombinant hexose and pentose transporter proteins. In one aspect is a recombinant xylose transporter protein. The recombinant xylose transporter protein includes a transporter motif sequence corresponding to amino acid residue positions 36, 37, 38, 39, 40, and 41 of Candida intermedia GXS1 protein. The transporter motif sequence has the sequence -G-G/F-X^-X^G-. X1 is D, C, G, H, I, L, or F. X2 is A, D, C, E, G, H, or I. X3 is N, C, Q, F, G, L, M, S, T, or P. In embodiments, the transporter motif sequence is not -G-G-L-I-F-G- or -G-G-F-I-F-G-.

[0056] X1 may be D, C, G, I, L, or F. X1 may be D, C, G, H, or F. X1 may be D. X1 may be C. X1 may be G. X1 may be I. X1 may be L. X1 may be H. X1 may be F. X2 may be D, C, E, G, H, or I. X2 may be E, G, H, or I. X2 may be H or I. X2 may be H. X2 may be I. X3 may be N, Q, F, M, S, T, or P. X3 may be F, M, S, or T. X3 may be S, T, or M. X3 may be T. X3 may be S. X3 may be M. When X1 is F, X2 may be I and X3 may be M or S.

[0057] The transporter motif sequence may be -G-G-F-I-M-G-, -G-F-F-I-M-G-, -G-G-F-I-S-G-, -G-F-F-I-S-G-, -G-G-F-I-T-G-, -G-F-F-I-T-G-, -G-G-F-L-M-G-, -G-F-F-L-M-G-, -G-G-F-L-S-G-, -G-F-F-L-S-G-, -G-G-F-L-T-G-, -G-F-F-L-T-G-, -G-G-F-H-M-G-, -G-F-F-H-M-G-, -G-G-F-H-S-G-, -G-F-F-H-S-G-, -G-G-F-H-T-G- or -G-F-F-H-T-G-. The transporter motif sequence may be -G-G-F-I-M-G-, -G-F-F-I-M-G-, -G-G-F-I-S-G-, -G-F-F-I-S-G-, -G-G-F-I-T-G-, or -G-F-F-I-T-G-. The transporter motif sequence may be -G-G-F-I-M-G-, -G-F-F-I-M-G-, -G-G-F-I-S-G-, or -G-F-F-I-S-G-. The transporter motif sequence may be -G-G-F-I-M-G-, or -G-F-F-I-M- G-. The transporter motif sequence may be -G-G-F-I-M-G-. The transporter motif sequence may be -G-F-F-I-M-G-. The transporter motif sequence may be -G-G-F-I-S-G-. The transporter motif sequence may be -G-F-F-I-S-G-. The transporter motif sequence may be -G-G-F-I-T-G-. The transporter motif sequence may be -G-F-F-I-T-G-. The transporter motif sequence may be -G-G-F-L-M-G-. The transporter motif sequence may be -G-F-F-L-M-G-. The transporter motif sequence may be -G-G-F-L-S-G-. The transporter motif sequence may be -G-F-F-L-S-G-. The transporter motif sequence may be -G-G-F-L-T-G-. The transporter motif sequence may be -G-F-F-L-T-G-. The transporter motif sequence may be -G-G-F-H-M-G-. The transporter motif sequence may be -G-F-F-H-M-G-. The transporter motif sequence may be -G-G-F-H-S-G-. The transporter motif sequence may be -G-F-F-H-S-G-. The transporter motif sequence may be -G-G-F-H-T-G-. The transporter motif sequence may be -G-F-F-H-T-G-.

[0058] The recombinant xylose transporter protein described herein may further include a mutation of an amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein. The amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein may be substituted with a Met, Ala, Ser, or Asn residue. The amino acid may be substituted with Met. The amino acid may be substituted with Ala. The amino acid may be substituted with Ser. The amino acid may be substituted with Asn. The recombinant xylose transporter protein may include a -G-G-F-I-M-G- transporter motif sequence and a Met substitution at the position corresponding to 297 of Candida intermedia GXS1 protein. The mutations of the amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein may prevent transport of hexoses by the recombinant xylose transporter. The mutations of the amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein, in combination with the transporter motif sequences described herein, may prevent transport of hexoses by the recombinant xylose transporter.

[0059] The recombinant xylose transporter protein may be derived from a sugar transporter protein (e.g. a transporter protein (e.g. a MFS transporter protein), a homolog thereof, or a functional fragment thereof, found in in a cell). The xylose transporter protein may be derived from a yeast cell transporter protein (e.g. a transporter protein, a homolog thereof, or a functional fragment thereof, found in in a yeast cell). The yeast cell transporter protein may be a MFS transporter protein. The yeast cell may be a species set forth in Figure 19 or Figure 20. The recombinant xylose transporter protein may be derived from a C. intermedia gxsl transporter protein (e.g. a gxsl transporter protein, a homolog thereof, or a functional fragment thereof,

found in C intermedia SEQ ID NO: 1), a S. stipitis rgt2 transporter protein (e.g. a rgt2 transporter protein, a homolog thereof, or a functional fragment thereof, found in S. stipitis), or a S.

cerevisiae hxt7 transporter protein (e.g. a hxt7 transporter protein, a homolog thereof, or a functional fragment thereof, found in 5*. cerevisiae). The recombinant xylose transporter protein may be derived from a C. intermedia gxsl transporter protein. The recombinant xylose transporter protein may be derived from a 5*. stipitis rgt2 transporter protein. The recombinant xylose transporter protein may be derived from a 5*. cerevisiae hxt7 transporter protein.

[0060] In another aspect is a recombinant galactose-arabinose transporter protein. The recombinant galactose-arabinose transporter protein includes a transporter motif sequence corresponding to residue positions 36, 37, 38, 39, 40, and 41 of Candida intermedia GXSl protein. The transporter motif sequence has the sequence -G-G/F-X4-X5-X6-G-. X4 is D, C, F, G, H, L, R, T, or P. X5 is A, C, E, F, H, K, S, P, or V. X6 is R, D, E, F, H, I, M, T, or Y. The sequence is not -G-G-L-V-Y-G-, or -G-G-F-V-F-G.

[0061] X4 may be D, F, G, L, R, or T. X4 may be R, T, H, or F. X4 may be R. X4 may be T. X4 may be H. X4 may be F. X5 may be A, E, F, P, H, or V. X5 may be P, H, or V. X5 may be P. X5 may be H. X5 may be V. X6 may be T, H, F, M, or Y. X6 may be F or Y. X6 may be T or M. X6 may be T. X6 may be H. X6 may be F. X6 may be M. X6 may be Y. When X4 is F or T, X5 may be P or I, and X6 may be M or T.

[0062] The transporter motif sequence may be -G-G-F-H-M-G-, -G-F-F-H-M-G-, -G-G-R-P-T-G-, -G-F-R-P-T-G-, -G-G-T-P-T-G-, or -G-F-T-P-T-G-. The transporter motif sequence may be -G-G-F-H-M-G-, -G-F-F-H-M-G-. The transporter motif sequence may be -G-G-R-P-T-G-, -G-F-R-P-T-G-. The transporter motif sequence may be -G-G-T-P-T-G-, or -G-F-T-P-T-G-. The transporter motif sequence may be -G-G-F-H-M-G-. The transporter motif sequence may be -G-F-F-H-M-G-. The transporter motif sequence may be -G-G-R-P-T-G-. The transporter motif sequence may be -G-F-R-P-T-G-. The transporter motif sequence may be -G-G-T-P-T-G-. The transporter motif sequence may be -G-F-T-P-T-G-.

[0063] The recombinant galactose-arabinose transporter protein described herein may include a mutation of an amino acid at the residue position corresponding to 297 of Candida intermedia GXSl protein. The amino acid at the residue position corresponding to 297 of Candida intermedia GXSl protein may be substituted with a Met, Thr, Ala, or He residue. The amino acid may be substituted with Met. The amino acid may be substituted with Thr. The amino acid may

be substituted with Ala. The amino acid may be substituted with He. The recombinant galactose-arabinose transporter protein may include a -G-G-T-P-T-G- transporter motif sequence and a Met substitution at the position corresponding to 297 of Candida intermedia GXS1 protein. The mutations of the amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein may prevent transport of hexoses, other than galactose, by the recombinant galactose-arabinose transporter. The mutations of the amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein, in combination with the transporter motif sequences described herein, may prevent transport of hexoses, other than galactose, by the recombinant galactose-arabinose transporter.

[0064] The recombinant galactose-arabinose transporter protein may be derived from a sugar transporter protein (e.g. a transporter protein (e.g. a MFS transporter protein), a homolog thereof, or a functional fragment thereof, found in in a cell). The recombinant galactose-arabinose transporter protein may be derived from a yeast cell transporter protein (e.g. a transporter protein, a homolog thereof, or a functional fragment thereof, found in in a yeast cell). The transporter protein may be a MFS transporter protein. The yeast cell may be a species set forth in Figure 19 or Figure 20. The recombinant galactose-arabinose transporter protein may be derived from a C intermedia gxsl transporter protein (e.g. a gxs l transporter protein, a homolog thereof, or a functional fragment thereof, found in C intermedia SEQ ID NO: 1), a S. stipitis rgt2 transporter protein (e.g. a rgt2 transporter protein, a homolog thereof, or a functional fragment thereof, found in S. stipitis), a S. cerevisiae hxtl transporter protein (e.g. a hxt7 transporter protein, a homolog thereof, or a functional fragment thereof, found in S. cerevisiae), or a S. cerevisiae GAL2 transporter protein (e.g. a GAL2 transporter protein, a homolog thereof, or a functional fragment thereof, found in 5*. cerevisiae). The recombinant galactose-arabinose transporter protein may be derived from a C. intermedia gxsl transporter protein. The recombinant galactose-arabinose transporter protein may be derived from a 5*. stipitis rgt2 transporter protein. The recombinant galactose-arabinose transporter protein may be derived from a 5*. cerevisiae hxt7 transporter protein. The recombinant galactose-arabinose transporter protein may be derived from a 5*. cerevisiae GAL2 transporter protein.

[0065] Further provided herein are nucleic acid sequences encoding the hexose or pentose transporter proteins described herein. In one aspect is a nucleic acid encoding a recombinant xylose transporter protein described herein. In another aspect is a nucleic acid encoding a recombinant galactose-arabinose transporter protein described herein. The nucleic acids may be RNA or DNA. The nucleic acids may be single- or double-stranded RNA or single- or double-stranded DNA. The nucleic acids may be located on a plasmid or other vector (e.g. a yeast artificial chromosome (YAC)). The nucleic acids may be introduced and expressed by a yeast cell using conventional techniques known to those in the art.

[0066] Provided herein are yeast cells that include a hexose or pentose transporter protein described herein. In one aspect is a yeast cell that includes a recombinant xylose transporter protein described herein. The yeast cell including a recombinant xylose transporter protein described herein may be a species as set forth in Figure 19 or Figure 20. The yeast cell including a recombinant xylose transporter protein described herein may be a S. stipitis yeast cell, a C. intermedia yeast cell, a 5*. cerevisiae yeast cell, a D. hansenii yeast cell, or a Y. Upolytica yeast cell. The yeast cell including a recombinant xylose transporter protein described herein may be capable of growth when placed in the presence of pentoses. The yeast cell including a recombinant xylose transporter protein described herein may be capable of growth, or have significantly increased growth compared to a yeast cell lacking the recombinant xylose transporter protein when placed in the presence of a xylose compound. The xylose compound is described herein. The xylose compound may be derived from lignocellulosic biomass.

[0067] The xylose compound may be present at a concentration of about 0.05 g/L to about 20 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 15 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 10 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 5 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 4 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 3 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 2 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 1 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 0.5 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 0.1 g/L. The xylose compound may be present at a concentration of about 0.05 g/L. The xylose compound may be present at a concentration of about 0.1 g/L. The xylose compound may be present at a concentration of about 0.5 g/L. The xylose compound may be present at a concentration of about 0.1 g/L. The xylose compound may be present at a concentration of about 0.5 g/L. The xylose compound may be present at a concentration of about 1 g/L. The xylose compound may be present at a concentration of about 2 g/L. The xylose compound may be present at a

concentration of about 3 g/L. The xylose compound may be present at a concentration of about 4 g/L. The xylose compound may be present at a concentration of about 5 g/L. The xylose compound may be present at a concentration of about 10 g/L. The xylose compound may be present at a concentration of about 15 g/L. The xylose compound may be present at a concentration of about 20 g/L.

[0068] The xylose compound may be present at a concentration of about 0.05 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 250 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 200 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 150 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 100 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 50 g/L. The xylose compound may be present at a concentration of about 0.05 g/L to about 25 g/L. The xylose compound may be present at a concentration of about 1 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 20 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 30 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 40 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 50 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 75 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 100 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 125 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 150 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 175 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 200 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 225 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 250 g/L to about 300 g/L. The xylose compound may be present at a concentration of about 275 g/L to about 300 g/L.

[0069] The xylose compound may be present at a concentration of about 10 g/L to about 275 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 250 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 225 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 200 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 175 g/L. The xylose

compound may be present at a concentration of about 10 g/L to about 150 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 125 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 100 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 75 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 50 g/L. The xylose compound may be present at a concentration of about 10 g/L to about 25 g/L.

[0070] The xylose compound may be present at a concentration of about 25 g/L. The xylose compound may be present at a concentration of about 50 g/L. The xylose compound may be present at a concentration of about 75 g/L. The xylose compound may be present at a concentration of about 100 g/L. The xylose compound may be present at a concentration of about 125 g/L. The xylose compound may be present at a concentration of about 150 g/L. The xylose compound may be present at a concentration of about 175 g/L. The xylose compound may be present at a concentration of about 200 g/L. The xylose compound may be present at a concentration of about 225 g/L. The xylose compound may be present at a concentration of about 250 g/L. The xylose compound may be present at a concentration of about 275 g/L. The xylose compound may be present at a concentration of about 300 g/L.

[0071] The yeast cell including a recombinant xylose transporter protein described herein may be incapable of growth, or have significantly impaired growth compared to a yeast cell lacking the recombinant xylose transporter protein when placed in the presence of only hexoses. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 20 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 15 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 10 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 5 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 4 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 3 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 2 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 1 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 0.5 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L to about 0.1 g/L. The hexose (e.g glucose) may be present at a concentration of about 0 05 g/L. The hexose (e.g. glucose) may be present at a concentration of about 0.1 g/L. The hexose (e.g. glucose) may be present at a concentration of about 0.5 g/L. The hexose (e.g. glucose) may be present at a concentration of about 0.1 g/L. The hexose (e.g. glucose) may be present at a concentration of about 0.5 g/L. The hexose (e.g. glucose) may be present at a concentration of about 1 g/L. The hexose (e.g. glucose) may be present at a concentration of about 2 g/L. The hexose (e.g. glucose) may be present at a concentration of about 3 g/L. The hexose (e.g. glucose) may be present at a concentration of about 4 g/L. The hexose (e.g. glucose) may be present at a concentration of about 5 g/L. The hexose (e.g. glucose) may be present at a concentration of about 10 g/L. The hexose (e.g. glucose) may be present at a concentration of about 15 g/L. The hexose (e.g.

glucose) may be present at a concentration of about 20 g/L.

[0072] The recombinant xylose transporter protein of the yeast cell may include a transporter motif sequence as set forth herein. The yeast cell may metabolize the xylose compound. The yeast cell may convert xylose compound to a biofuel (e.g. ethanol) or a biochemical described herein. The yeast cell may convert xylose compound to a biofuel (e.g. ethanol). The yeast cell may convert xylose compound to a biochemical described herein.

[0073] In another aspect is a yeast cell that includes a recombinant galactose-arabinose transporter protein described herein. The yeast cell including a recombinant galactose-arabinose transporter protein described herein may be a species as set forth in Figure 19 or Figure 20. The yeast cell including the recombinant galactose-arabinose transporter protein may be a 5*. stipitis yeast cell, a C. intermedia yeast cell, a 5*. cerevisiae yeast cell, a D. hansenii yeast cell, or a Y. Upolytica yeast cell. The yeast cell including the recombinant galactose-arabinose transporter protein may be capable of growth, or have significantly increased growth compared to a yeast cell lacking the recombinant galactose-arabinose transporter protein when placed in the presence of pentoses (e.g. arabinose). The yeast cell including the recombinant galactose-arabinose transporter protein may be capable of growth, or have significantly increased growth compared to a yeast cell lacking the recombinant galactose-arabinose transporter protein when placed in the presence of an arabinose compound. The arabinose compound is described herein. The arabinose compound may be derived from lignocellulosic biomass.

[0074] The arabinose compound may be present at a concentration of about 0.05 g/L to about 20 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 15 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 10 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 5 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 4 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 3 g/L. The

arabinose compound may be present at a concentration of about 0.05 g/L to about 2 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 1 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 0.5 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 0.1 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L. The arabinose compound may be present at a concentration of about 0.1 g/L. The arabinose compound may be present at a concentration of about 0.5 g/L. The arabinose compound may be present at a concentration of about 0.1 g/L. The arabinose compound may be present at a concentration of about 0.5 g/L. The arabinose compound may be present at a concentration of about 1 g/L. The arabinose compound may be present at a concentration of about 2 g/L. The arabinose compound may be present at a concentration of about 3 g/L. The arabinose compound may be present at a concentration of about 4 g/L. The arabinose compound may be present at a concentration of about 5 g/L. The arabinose compound may be present at a concentration of about 10 g/L. The arabinose compound may be present at a concentration of about 15 g/L. The arabinose compound may be present at a concentration of about 20 g/L.

[0075] The arabinose compound may be present at a concentration of about 0.05 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 250 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 200 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 150 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 100 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 50 g/L. The arabinose compound may be present at a concentration of about 0.05 g/L to about 25 g/L. The arabinose compound may be present at a concentration of about 1 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 20 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 30 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 40 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 50 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 75 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 100 g/L to about 300 g/L.

The arabinose compound may be present at a concentration of about 125 g/L to about 300 g/L.

The arabinose compound may be present at a concentration of about 150 g/L to about 300 g/L.

The arabinose compound may be present at a concentration of about 175 g/L to about 300 g/L.

The arabinose compound may be present at a concentration of about 200 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 225 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 250 g/L to about 300 g/L. The arabinose compound may be present at a concentration of about 275 g/L to about 300 g/L.

[0076] The arabinose compound may be present at a concentration of about 10 g/L to about 275 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 250 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 225 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 200 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 175 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 150 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 125 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 100 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 75 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 50 g/L. The arabinose compound may be present at a concentration of about 10 g/L to about 25 g/L.

[0077] The arabinose compound may be present at a concentration of about 25 g/L. The arabinose compound may be present at a concentration of about 50 g/L. The arabinose compound may be present at a concentration of about 75 g/L. The arabinose compound may be present at a concentration of about 100 g/L. The arabinose compound may be present at a concentration of about 125 g/L. The arabinose compound may be present at a concentration of about 150 g/L. The arabinose compound may be present at a concentration of about 175 g/L. The arabinose compound may be present at a concentration of about 200 g/L. The arabinose compound may be present at a concentration of about 225 g/L. The arabinose compound may be present at a concentration of about 250 g/L. The arabinose compound may be present at a concentration of about 275 g/L. The arabinose compound may be present at a concentration of about 300 g/L.

[0078] The yeast cell including the recombinant galactose-arabinose transporter protein may be incapable of growth, or have significantly impaired growth compared to a yeast cell lacking the recombinant galactose-arabinose transporter protein when placed in the presence of hexoses such as glucose or mannose (i.e. the recombinant galactose-arabinose transporter protein does not transport glucose or mannose). The hexose (e.g. glucose) may in present in a concentration as set forth herein. The yeast cell including the recombinant galactose-arabinose transporter protein may be capable of growth, or have significantly increased growth compared to a yeast cell lacking the recombinant galactose-arabinose transporter protein when placed in the presence of a galactose compound. The galactose compound is described herein. The galactose compound may be derived from lignocellulosic biomass.

[0079] The galactose compound may be present at a concentration of about 0.05 g/L to about 20 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 15 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 10 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 5 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 4 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 3 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 2 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 1 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 0.5 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 0.1 g/L. The galactose compound may be present at a concentration of about 0.05 g/L. The galactose compound may be present at a concentration of about 0.1 g/L. The galactose compound may be present at a concentration of about 0.5 g/L. The galactose compound may be present at a concentration of about 0.1 g/L. The galactose compound may be present at a concentration of about 0.5 g/L. The galactose compound may be present at a concentration of about 1 g/L. The galactose compound may be present at a concentration of about 2 g/L. The galactose compound may be present at a concentration of about 3 g/L. The galactose compound may be present at a concentration of about 4 g/L. The galactose compound may be present at a concentration of about 5 g/L. The galactose compound may be present at a concentration of about 10 g/L. The galactose compound may be present at a concentration of about 15 g/L. The galactose compound may be present at a concentration of about 20 g/L.

[0080] The galactose compound may be present at a concentration of about 0.05 g/L to about 300 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 250 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 200 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 150 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 100 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 50 g/L. The galactose compound may be present at a concentration of about 0.05 g/L to about 25 g/L. The galactose compound may be present at a concentration of about 1 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 10 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 20 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 30 g/L to about 300 g/L. The galactose compound may be present at a concentration of about 40 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 50 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 75 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 100 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 125 g/L to about 300 g/L. The galactose compound may be present at a concentration of about 150 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 175 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 200 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 225 g/L to about 300 g/L.

The galactose compound may be present at a concentration of about 250 g/L to about 300 g/L. The galactose compound may be present at a concentration of about 275 g/L to about 300 g/L.

[0081] The galactose compound may be present at a concentration of about 10 g/L to about 275 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 250 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 225 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 200 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 175 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 150 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 125 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 100 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 75 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 50 g/L. The galactose compound may be present at a concentration of about 10 g/L to about 25 g/L.

[0082] The galactose compound may be present at a concentration of about 25 g/L. The galactose compound may be present at a concentration of about 50 g/L. The galactose compound may be present at a concentration of about 75 g/L. The galactose compound may be present at a concentration of about 100 g/L. The galactose compound may be present at a concentration of about 125 g/L. The galactose compound may be present at a concentration of about 150 g/L. The galactose compound may be present at a concentration of about 175 g/L. The galactose

compound may be present at a concentration of about 200 g/L. The galactose compound may be present at a concentration of about 225 g/L. The galactose compound may be present at a concentration of about 250 g/L. The galactose compound may be present at a concentration of about 275 g/L. The galactose compound may be present at a concentration of about 300 g/L.

[0083] The yeast cell including the recombinant galactose-arabinose transporter protein may be capable of growth, or have significantly increased growth when compared to a yeast cell lacking the recombinant galactose-arabinose transporter protein when placed in the presence of an arabinose compound and a galactose compound. The arabinose compound is described herein and may be present in a concentration described herein. The galactose compound is described herein and may be present in a concentration described herein. The arabinose compound may be derived from lignocellulosic biomass. The galactose compound may be derived from

lignocellulosic biomass.

[0084] The recombinant galactose-arabinose transporter protein of the yeast cell may include a transporter motif sequence as set forth herein. The yeast cell may metabolize the arabinose compound. The yeast cell may metabolize the galactose compound. The yeast cell may convert the arabinose compound to a biofuel (e.g. ethanol) or a biochemical described herein. The yeast cell may convert the galactose compound to a biofuel (e.g. ethanol) or a biochemical described herein. The yeast cell may convert the arabinose compound to a biofuel (e.g. ethanol). The yeast cell may convert the arabinose compound to a biochemical described herein. The yeast cell may convert the galactose compound to a biofuel (e.g. ethanol). The yeast cell may convert the galactose compound to a biochemical described herein.

[0085] Also provided herein are methods of transporting hexose or pentose moieties into a yeast cell. In one aspect is a method for transporting xylose into a yeast cell. The method includes contacting a yeast cell having a recombinant xylose transport protein described herein with a xylose compound described herein. The recombinant xylose transport protein is allowed to transport the xylose compound into the cell. The yeast cell may be a yeast cell described herein. The yeast cell may be a 5*. stipitis yeast cell, a C. intermedia yeast cell, a 5*. cerevisiae yeast cell, a D. hansenii yeast cell, or a Y. Upolytica yeast cell.

[0086] The xylose compound may be derived from lignocellulosic biomass, hemicellulose, or xylan. The xylose compound may be derived from lignocellulosic biomass. The xylose compound may be derived from hemicellulose. The xylose compound may be derived from

xylan. The yeast cell may metabolize the xylose compound. The yeast cell may preferentially grow in the presence of a xylose compound and may not grow using only another sugar source (e.g. glucose) when compared to a yeast cell lacking the recombinant xylose transporter protein. The xylose compound may be present in a concentration described herein. The yeast cell may convert the xylose compound to a biofuel (e.g. ethanol) or to a biochemical described herein. The yeast cell may convert the xylose compound to a biofuel (e.g. ethanol). The yeast cell may convert the xylose compound to a biochemical described herein.

[0087] The recombinant xylose transport protein may have a binding affinity of about 1 mM to about 0.02 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of about 0.8 mM to about 0.02 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of 0.8 mM to about 0.05 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of 0.8 mM to about 0.1 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of 0.8 mM to about 0.2 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of 0.8 mM to about 0.3 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of 0.8 mM to about 0.4 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of 0.8 mM to about 0.5 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of 0.8 mM to about 0.6 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of 0.8 mM to about 0.7 mM for a xylose compound.

[0088] The recombinant xylose transport protein may have a binding affinity of at least 0.02 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.05 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.1 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.2 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.3 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.4 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.5 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.6 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.7 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.8 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 0.9 mM for a xylose compound. The recombinant xylose transport protein may have a binding affinity of at least 1 mM for a xylose compound.

[0089] The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 7 nmol min"1 gDCW"1 to about 15 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 8 nmol min"1 gDCW"1 to about 15 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 9 nmol min"1 gDCW"1 to about 15 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 15 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 1 1 nmol min"1 gDCW"1 to about 15 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 12 nmol min ^ gDCW"1 to about 15 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 13 nmol min"1 gDCW"1 to about 15 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 14 nmol min"1 gDCW"1 to about 15 nmol min"1 gDCW"1.

[0090] The recombinant xylose transport protein may have a rate of at least 7 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 8 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 9 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 10 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 1 1 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 12 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 13 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 14 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 15 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell.

[0091] The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 20 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 30 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 40 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 50 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 60 nmol min"1 gDCW"1 to about 70 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 80 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 90 nmol min ^ gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 100 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 1 10 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 120 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 130 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 140 nmol min"1 gDCW"1 to about 150 nmol min"1 gDCW"1.

[0092] The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 140 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 130 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 120 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 1 10 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 100 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 90 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 80 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min 1 gDCW"1 to about 70 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 60 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 50 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 40 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 30 nmol min"1 gDCW"1. The recombinant xylose transport protein may have a rate of transporting a xylose compound into a yeast cell of about 10 nmol min"1 gDCW"1 to about 20 nmol min"1 gDCW"1.

[0093] The recombinant xylose transport protein may have a rate of at least 20 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 30 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 40 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 50 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 60 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 70 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 80 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 90 nmol min 1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 100 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 110 nmol min"1 gDCW" 1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 120 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 130 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 140 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell. The recombinant xylose transport protein may have a rate of at least 150 nmol min"1 gDCW"1 of transporting a xylose compound into a yeast cell.

[0094] In another aspect is a method of transporting galactose or arabinose into a yeast cell. The method includes contacting a yeast cell including a recombinant galactose-arabinose transport protein described herein, with a galactose compound or an arabinose compound described herein. The recombinant galactose-arabinose transport protein is allowed to transport the galactose compound or the arabinose compound into the yeast cell. The yeast cell may be a yeast cell described herein. The yeast cell may be a S. stipitis yeast cell, a C. intermedia yeast cell, a 5*. cerevisiae yeast cell, a D. hansenii yeast cell, or a Y. Upolytica yeast cell.

[0095] In the presence of arabinose, the recombinant galactose-arabinose transport protein may transport arabinose into a yeast cell. The arabinose compound may be present at a concentration as set forth herein. The arabinose compound may be derived from lignocellulosic biomass, hemicellulose, or arabinoxylan. The arabinose compound may be derived from lignocellulosic biomass. The arabinose compound may be derived from hemicellulose. The arabinose compound may be derived from arabinoxylan. The yeast cell may metabolize the arabinose compound. The yeast cell may preferentially grow in the presence of an arabinose compound and may not grow using only another sugar source (e.g. glucose) as compared to a yeast cell lacking the recombinant galactose-arabinose transporter protein. The yeast cell may convert the arabinose compound to a biofuel (e.g. ethanol) or to a biochemical (e.g. an organic acid.) The yeast cell may convert the arabinose compound to a biofuel (e.g. ethanol). The yeast cell may convert the arabinose compound to a biochemical described herein.

[0096] In the presence of galactose, the recombinant galactose-arabinose transport protein transports galactose into a yeast cell. The galactose compound may be at a concentration as set forth herein. The galactose compound may be derived from lignocellulosic biomass,

hemicellulose, or galactan. The galactose compound may be derived from lignocellulosic

biomass. The galactose compound may be derived from hemicellulose. The galactose compound may be derived from galactan. The yeast cell may metabolize the galactose compound. The yeast cell may preferentially grow in the presence of a galactose compound and may not grow using only another sugar source (e.g. glucose). The yeast cell may convert the galactose compound to a biofuel (e.g. ethanol) or to a biochemical described herein. The yeast cell may convert the galactose compound to a biofuel (e.g. ethanol). The yeast cell may convert the galactose compound to a biochemical described herein.

I. Embodiments

[0097] Embodiment 1 A recombinant xylose transporter protein comprising a transporter motif sequence corresponding to amino acid residue positions 36, 37, 38, 39, 40, and 41 of Candida intermedia GXS1 protein, wherein said transporter motif sequence is -G-G/F-X1-X2-X3-G-; wherein, X1 is D, C, G, H, I, L, or F; X2 is A, D, C, E, G, H, or I; X3 is N, C, Q, F, G, L, M, S, T, or P; and wherein, said transporter motif sequence is not -G-G-L-I-F-G- or -G-G-F-I-F-G-.

[0098] Embodiment 2 The recombinant xylose transporter protein of embodiment 1, wherein, X1 is D, C, G, H, or F; X2 is H or I; and X3 is S, T, or M.

[0099] Embodiment 3 The recombinant xylose transporter protein of embodiment 1 or 2, wherein X1 is F, X2 is I, and X3 is M or S.

[0100] Embodiment 4 The recombinant xylose transporter protein of any one of embodiments 1 to 3, wherein said transporter motif sequence is -G-G-F-I-M-G-, -G-F-F-I-M-G-, -G-G-F-I-S-G-, -G-F-F-I-S-G-, -G-G-F-I-T-G-, -G-F-F-I-T-G-, -G-G-F-L-M-G-, -G-F-F-L-M-G-, -G-G-F-L-S-G-, -G-F-F-L-S-G-, -G-G-F-L-T-G-, -G-F-F-L-T-G-, -G-G-F-H-M-G-, -G-F-F-H-M-G-, -G-G-F-H-S-G-, -G-F-F-H-S-G-, -G-G-F-H-T-G- or -G-F-F-H-T-G-.

[0101] Embodiment 5 The recombinant xylose transporter protein of any one of embodiments 1 to 4, wherein said transporter motif sequence is -G-G-F-I-M-G-, -G-F-F-I-M-G-, -G-G-F-I-S-G-, or -G-F-F-I-S-G-.

[0102] Embodiment 6 The recombinant xylose transporter protein of any one of embodiments 1 to 5 further comprising a mutation of an amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein.

[0103] Embodiment 7 The recombinant xylose transporter protein of any one of embodiments 1 to 6, wherein said amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein is substituted with a Met, Ala, Ser, or Asn residue.

[0104] Embodiment 8 The recombinant xylose transporter protein of any one of embodiments 1 to 7, wherein said recombinant xylose transporter protein is derived from a C intermedia gxsl transporter protein, a S. stipitis rgt2 transporter protein, or a S. cerevisiae hxtl transporter protein.

[0105] Embodiment 9 A recombinant galactose-arabinose transporter protein comprising a transporter motif sequence corresponding to amino acid residue positions 36, 37, 38, 39, 40, and 41 of Candida intermedia GXS1 protein, wherein said transporter motif sequence is -G-G/F-X4-X5-X6-G-; wherein, X4 is D, C, F, G, H, L, R, T, or P; X5 is A, C, E, F, H, K, S, P, or V; X6 is R, D, E, F, H, I, M, T, or Y; and wherein said sequence is not -G-G-L-V-Y-G-, or -G-G-F-V-F-G-.

[0106] Embodiment 10 The recombinant galactose-arabinose transporter protein of embodiment 9, wherein, X4 is R, T, H, or F; X5 is P, H, or V; and X6 is T, H, F, M, or Y.

[0107] Embodiment 1 1 The recombinant galactose-arabinose transporter protein of embodiment 9, wherein X4 is F or T, X5 is P or I, and X6 is M or T.

[0108] Embodiment 12 The recombinant galactose-arabinose transporter protein of embodiment 10 or 11, wherein said transporter motif sequence is -G-G-F-H-M-G-, -G-F-F-H-M-G-, -G-G-R-P-T-G-, -G-F-R-P-T-G-, -G-G-T-P-T-G-, or -G-F-T-P-T-G-.

[0109] Embodiment 13 The recombinant galactose-arabinose transporter protein of any one of embodiments 9 to 12, wherein said galactose-arabinose transporter protein further comprises a mutation of an amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein.

[0110] Embodiment 14 The recombinant galactose-arabinose transporter protein of any one of embodiments 9 to 13, wherein said amino acid at the residue position corresponding to 297 of Candida intermedia GXS1 protein is substituted with a Met, Thr, Ala, or He residue.

[0111] Embodiment 15 The recombinant galactose-arabinose transporter protein of any one of embodiments 9 to 14, wherein said recombinant galactose-arabinose transporter protein is derived from a C intermedia gxsl transporter protein, a S. stipitis rgt2 transporter protein, a S. cerevisiae hxtl transporter protein, or a S. cerevisiae GAL2 protein.

[0112] Embodiment 16 A yeast cell comprising the recombinant xylose transporter protein of any one of embodiments 1 to 8.

[0113] Embodiment 17 A yeast cell comprising the recombinant galactose-arabinose transporter protein of any one of embodiments 9 to 15.

[0114] Embodiment 18 A nucleic acid encoding the recombinant xylose transporter protein of any one of embodiments 1 to 8.

[0115] Embodiment 19 A nucleic acid encoding the recombinant galactose-arabinose transporter protein of any one of embodiments 9 to 15.

[0116] Embodiment 20 A method of transporting xylose into a yeast cell, said method comprising: contacting a yeast cell comprising the recombinant xylose transporter protein of any one of embodiments 1 to 8 with a xylose compound; and allowing said recombinant xylose transporter protein to transport said xylose compound into said yeast cell.

[0117] Embodiment 21 The method of embodiment 20, wherein said xylose compound forms part of lignocellulosic biomass, hemicellulose, or xylan.

[0118] Embodiment 22 The method of embodiment 20 or 21, wherein said yeast cell metabolizes said xylose compound.

[0119] Embodiment 23 The method of any one of embodiments 20 to 22, wherein said yeast cell converts said xylose compound to a biofuel.

[0120] Embodiment 24 The method of any one of embodiments 20 to 23, wherein said recombinant xylose transporter protein has a binding affinity of at least 0.7 mM for said xylose compound.

[0121] Embodiment 25 The method of any one of embodiments 20 to 24, wherein said recombinant xylose transporter protein has a rate of at least 15 nmol min 1 gDCW"1 of transporting said xylose compound into said yeast cell.

[0122] Embodiment 26 A method of transporting galactose or arabinose into a yeast cell, said method comprising: contacting a yeast cell comprising the recombinant galactose-arabinose transporter protein of any one of embodiments 9 to 15 with a galactose compound or an arabinose compound; and allowing said recombinant galactose-arabinose transporter protein to transport said galactose compound or said arabinose compound into said yeast cell.

[0123] Embodiment 27 The method of embodiment 26, wherein said recombinant galactose-arabinose transporter protein is contacted with an arabinose compound.

[0124] Embodiment 28 The method of any one of embodiments 26 to 27, wherein said arabinose compound forms part of lignocellulosic biomass, hemicellulose or arabinoxylan.

[0125] Embodiment 29 The method of any one of embodiments 26 to 28, wherein said yeast cell metabolizes said arabinose compound.

[0126] Embodiment 30 The method of any one of embodiments 26 to 29, wherein said yeast cell converts said arabinose compound to a biofuel.

[0127] Embodiment 31 The method of any one of embodiments 26 to 30, wherein said recombinant galactose-arabinose transporter protein is contacted with a galactose compound.

[0128] Embodiment 32 The method of any one of embodiments 26 to 31, wherein said galactose compound forms a part of lignocellulosic biomass, hemicellulose, or galactan.

[0129] Embodiment 33 The method of any one of embodiments 26 to 32, wherein said yeast cell metabolizes said galactose compound.

[0130] Embodiment 34 The method of any one of embodiments 26 to 33, wherein said yeast cell converts said galactose compound to a biofuel.

[0131] Embodiment 35 The method of embodiment 20, wherein said yeast cell is a S. stipitis yeast cell, a C. intermedia yeast cell, a 5*. cerevisiae yeast cell, a D. hansenii yeast cell, or a Y. iipoiytica yeast cell.

[0132] Embodiment 36 The method of embodiment 26, wherein said yeast cell is a S. stipitis yeast cell, a C. intermedia yeast cell, a 5*. cerevisiae yeast cell, a D. hansenii yeast cell, or a Y. iipoiytica yeast cell.

II. Examples

[0133] Example 1 : Identification of the G-G/F-XXX-G motif that controls sugar transport preference.

[0134] A multiple sequence alignment of 26 previously cloned transporters (36) indicates that Phe40 was part of a highly conserved glycine-rich motif of the form G-G/F-XXX-G, where X represents a variable, but usually nonpolar amino acid residue. In C. intermedia GXSl, the wild

type motif is G G V L F G . The high conservation of this motif suggested it could be responsible for xylose uptake, transporter efficiency, and monosaccharide selectivity. To further corroborate this hypothesis, an additional 20 putative transporters were identified using a BLAST search seeded with transporters functionally characterized in 5*. cerevisiae EX.12, a recombinant strain lacking endogenous monosaccharide transporters (Figure 8 and Table 1) (26, 38). The vast majority of these transporters were functional and all possessed a similar motif. Among these transporters, D. hansenii 2D01474 confers much faster growth on xylose than on glucose and S. stipitis RGT2 confers the fastest growth on xylose of all the S. stipitis derived transporters in this study.

[0135] Following the functional characterization, motif sequence was correlated with transporter carbon source growth profile. Four major phenotypic classifications were made: (a) transporters that failed to function heterologously (μ¾11 = 0), (b) transporters that conferred growth on a hexose but not xylose (μχ = 0), (c) transporters that conferred growth on xylose but not as fast as glucose (μχ < μο), and (d) transporters that conferred a higher growth rate on xylose than on glucose (μχ > μο)· Figure 1A displays the relative proportions of each of these classifications in the group of 46 transporters studied. To characterize the sequence, four major motif classifications were made: (a) a full G-G/F-XXXG motif, (b) a related S-G-XXXG motif, (c) a motif unrelated to the glycine rich motif, and (d) the lack of homology to other transporters at both the motif and surrounding residues. Figure IB depicts the distribution of the four sequence motif classifications within the four phenotypic classifications. Strikingly, there is a clear enrichment of the G-G/F-XXXG motif among the functional transporters that enable high xylose transport rates. In fact, this motif is exclusively seen in phenotype class (d) where μχ > μο· The enrichment and convergence of the variable residues within the motif is displayed in Figure 1C. It should be noted that the consensus sequence from this analysis appears to be G-G/F-XX-F-G. Yet, variations at the consensus F residue led to the discovery of the motif, therefore this position was considered variable. Figure 1C highlights the strong correlation between sequence motif and xylose transport function and suggests an important role of TMS 1 on sugar recognition.

[0136] Identification of potentiating variable residues within the G-G/F-X-X-X-G motif.

[0137] To examine the role of the variable region, complete saturation mutagenesis was performed for each of the three residues (Val38, Leu39, and Phe40) in C. intermedia GXS1 and evaluated the impact on carbon source growth profile as measured by growth rate. Previous studies demonstrate that growth rate in this test strain is a good surrogate for transporter kinetics (36, 38). Specifically, the fractional change in growth rate of S. cerevisiae EX.12 on glucose, xylose, galactose, fructose, or mannose as the sole carbon source was evaluated compared to the wild-type transporter. The impact of each residue can be classified as having no change, altered efficiency, altered selectivity, or a combination of the three (Figure 2). For creating xylose specific transporters, the goal is to identify mutations that attenuate hexose growth while either amplifying or maintaining xylose growth.

[0138] Members of the C. intermedia gxs l Val38 saturation library (Figure 3 A) display differential exponential growth rates with the most significant one being the Phe38 substitution. This mutant confers a selectivity phenotype that almost completely attenuates glucose exponential growth rate while amplifying exponential xylose growth rate by 50%. Other substitutions that confer desirable selectivity phenotypes are Asp , Cys , Gly , and His . All of these affect the growth profile in different patterns, but none as significantly as Phe38. Three

38 38 38

substitutions, He , Leu , and Met , differentially amplify growth on multiple sugars while glucose growth remains unchanged. The Leu38 substitution in particular increases exponential xylose growth rate by 73% without altering glucose exponential growth rate significantly. Ala38 attenuates growth on glucose only. Nearly all of the remaining substitutions attenuate growth, yet many preferentially attenuate growth on hexoses. In this subset, Lys38 attenuates growth on glucose, fructose, and mannose without affecting growth rate on xylose. The frequency of selectivity and differentially attenuating phenotypes arising at this residue indicates that position 38 predominately influences monosaccharide selectivity.

[0139] Table 1: Exponential growth rate values for each cloned transporter


[0140] Nearly all members of the Leu saturation library (Figure 3B) display uniform attenuation patterns across sugars. Thus, residue 39 appears to greatly control transporter efficiency. Nevertheless, several of these substitutions differentially attenuate growth.

Specifically, Asp , Cys , Gly , His , He , and Phe reduce exponential growth on hexoses without drastically altering xylose growth rate. Of these, His39 and He39 establish the greatest difference between the hexose and pentose growth rates.

[0141] Members of the Phe library (Figure 3C) display differential carbon source selectivity similar to Val38 and have the greatest frequency of selectivity substitutions. Specifically, amino acid substitutions that confer a selectivity phenotype for xylose over glucose are Asn40, Cys40, Gly40, Leu40, Met40, Ser40, and Thr40. Of these, Ser40 and Met40 appear as the most significant. There are several attenuating substitutions that can be seen at residue 40 including Arg40, Asp40, Glu40, He40, Lys40, Pro40, and Tyr40. Of these, Pro40 appears as the only one that does not attenuate growth on xylose. Finally, Ala40, His40, and Trp40 confer increased growth on most of the monosaccharides tested. In summary, residues 38 and 40 appear to play a role in transporter selectivity while residue 39 appears to play a role for controlling net transporter efficiency. In general, hydrophobic residues of moderate to large size were beneficial for xylose growth, while charged residues were not (also seen with the evaluated transporters in Figure 1C). These motif design guidelines may be used to reprogram transporter function.

[0142] Rewiring C. intermedia GXSl into a xylose specific transporter.

[0143] Using the design guidelines discovered above, triple mutants were constructed to investigate the synergy between xylose favoring substitutions (in particular, Phe38, He39, and Ser40 / Met40). Both Phe38 He39 Ser40 and Phe38 He39 Met40 attenuate glucose exponential growth while maintaining or slightly increasing xylose exponential growth (Figure 4A), with the Phe38 He39 Met40 triple mutant attenuating glucose growth to the same level as the negative control. Average growth curves on xylose and glucose (Figure 4B-C) highlight that both triple mutants maintain wild-type xylose growth profile while severely attenuate glucose growth. Further characterization of the best mutant, gxsl Phe38 He39 Met40 was performed. First, to assay transport capacity, high cell density fermentations with xylose and glucose were performed (Figure 5A-B). The Phe38 He39 Met40 triple mutant displayed no appreciable glucose uptake whereas xylose uptake has become more efficient compared to the wild-type GXSl. These results display a rewiring of the sugar uptake ratio. However, despite minimizing glucose transport capacity, glucose at levels of 5 g/L still appear to inhibit xylose growth (Figure 5C). This finding is corroborated by high cell density cofermentations (Figure 9 and Figure 10).

[0144] Radiolabeled xylose uptake experiments were performed to quantify the improvement of transport kinetics in the Phe38 He39 Met40 triple mutant. The improvements in xylose utilization observed at high cell density culturing were mainly due to a doubling in Vmax (Figure 5D). An increased KM was observed as well (Figure 5E), a phenotype observed in previous efforts to engineer this transporter (38). Nevertheless, the binding affinity is still quite high for practical culturing at a value corresponding to around 0.1 g/L (Table 2). These kinetics experiments were also performed in the presence of glucose and no radiolabelled xylose uptake was detected indicating that while glucose cannot pass through the transporter, it can still bind and inhibit xylose uptake. Hence, binding appears to occur at a different residue.

[0145] Table 2: kinetics values calculated from radiolabeled xylose uptake

Gem m p414«TEF KM

(mM) (nrnol mirf gDCW" }

C,l« GXSI 0.0256 ± 0.0659 7.23 ± 0.6

CI GXSI Fmi M 0.721 ± 0.1 16 15.01 ± 2.36

[0146] The G-G/F-XXXG motif can be used to rewire other transporters

[0147] To test how broad these design guidelines are for transporters, the conserved G-G/F-XXXG motif was utilized to reengineer the sugar preference of other predominately hexose transporters. Specifically, two transporters, S. stipitis RGT2 and S. cerevisiae HXT7, were selected based on evolutionary distance from GXSI. S. stipitis RGT2 is closely related to C. intermedia GXSI, while the native HXT transporters are more distant (Figures 11 and 12). First, the impact of rewiring the closely related transporter, S. stipitis RGT2 was investigated. This transporter contains a G36G37I38L39F40G41 motif and two separate point mutations were characterized, Phe38 and Met40. In both cases, glucose growth has been completely attenuated (Figure 6). Most striking is the Met40 mutation, which eliminates growth on all carbon sources but xylose and galactose. By modifying the motif in RGT2, two additional mutant proteins were generated that transport xylose, but not glucose.

[0148] Second, the potential to rewire S. cerevisiae HXT7, a more distantly related protein yet is able to efficiently transport hexoses and xylose in yeast, was evaluated (32, 42). Given the proficiency of hexose transport by this protein, rewiring to attenuate growth on hexoses presents a greater challenge. The native motif within 5*. cerevisiae HXT7 is G36G37F38V39F40G41. Two double mutations to this motif - Ile39Met40 and His39Met40 were initially evaluated. Figure 7 demonstrates that the Ile39Met40 double mutant amplified xylose exponential growth and attenuated growth on all hexoses save glucose whereas the His39Met40 double mutant attenuated glucose growth yet also severely attenuated xylose exponential growth. Previous studies have indicated that mutations at Asp340 can eliminate glucose transport (39) in HXT7 and transport of nearly all monosaccharides is severely attenuated with this mutation was verify herein (Figure 7).

Coupling the Met mutation with the He Met double mutant resulted in robust growth on xylose while maintaining the inability to transport glucose. With this triple mutant, a robust hexose transporter was converted to a xylose transporter unable to support growth on glucose.

[0149] Thus, a short, six residue motif of the form G-G/F-XXXG in TMS 1 was identified that exerts control over selectivity and efficiency of monosaccharide transport of MFS family transporters. This motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Altering the composition of the variable region changes the sugar uptake profiles of these transporters and can thus be used to rewire transporter function. Altering the residues in this domain can eliminate glucose transport while retaining xylose transport, a major step forward for molecular transporter engineering. As a result, several transporter mutants were create that support the transport of xylose and not glucose.

[0150] Hydrophobic, nonpolar, and moderate to large size residues often attenuated glucose compared to xylose. Amino acids such as Phe, He, Ser, and Met were among the most effective substitutions that differentially amplified xylose growth rate. While many of these residues are found naturally in wild type motif sequences (Figure 2), the combinations found herein

(particularly Phe38 He39 Met40) are not found naturally. Hypotheses concerning transporter substrate recognition and transport mechanism may be formed based on these results. Without being bound to any particular theory, the advantage of large and nonpolar residues suggests that glucose growth attenuation is due to steric exclusion. The larger side chains may physically restrict the size of the pore, allowing the smaller xylose molecule to bind and traverse more efficiently than larger hexoses. A similar hypothesis has been proposed to explain an observed correlation between amino acid size and transporter function for glucose (43). This hypothesis is supported by the crystal structure of a related MFS transporter, E. coli xylE (41). Based on the structure, E. coli xylE Phe24, an analogous residue to C. intermedia gxsl Phe40, appears to interact with sugars as they pass through the pore. E. coli xylE is too dissimilar from yeast MFS transporters to enable structure prediction, yet this evidence suggests that this residue appears to play a role in all MFS sugar transporters.

[0151] Transporters from Neurospora crassa and S. stipitis were found to be exclusive for xylose in uptake assays (35), but are unable to support robust growth of recombinant S.

cerevisiae on xylose. The Escherichia coli xylE transporter is xylose specific when expressed in its native host (44), but is inhibited by glucose and remains non-functional in S. cerevisiae despite attempts at directed evolution. Prior to this work, no evidence has demonstrated a defined transporter engineering approach that is able to effectively eliminate glucose transport while amplifying xylose transport and supporting robust xylose growth. The mutants generated in this study demonstrate this desirable phenotype and provide evidence that the G-G/F-XXXG motif controls transport phenotype in a large number of MFS transport proteins.

[0152] It is also important to note that altering this motif in C. intermedia GXS 1 not only had an impact on glucose uptake, but also had an impact on the kinetics of xylose uptake.

Specifically, the KM for xylose was significantly increased compared to wild type, indicating that exclusion of glucose was obtained at the expense of reduced affinity for xylose. Nevertheless, the affinity for xylose remains sufficiently high for nearly all fermentation conditions (KM = 0.721 ± 0.1 16 mM, or approximately 0.1 g/L), and was partially compensated by a doubling in Vmax (Figure 5). This result suggests a complex set of interactions between the transporter and sugar substrate, and is similar to other mutants of C. intermedia GXS1 (38).

[0153] In the course of identifying and validating this motif, several novel native and heterologous transporters were identified and shown to possess previously unreported phenotypes (Figure 13). The transporter D. hansenii 2D01474 can natively support growth on xylose compared with glucose. The transporter S. stipitis RGT2 confers the fastest growth rate on xylose over any ORF cloned from S. stipitis. Both of these transporters are closely related to C. intermedia GXS1 (Figure 12) and may present a new class of related transporters that make excellent starting scaffolds for engineering exclusive xylose uptake. Of the remaining novel ORFs studied here, one group (D. hansenii 2E01166, D. hansenii 2B05060, S. cerevisiae STL1, and S. stipitis AUT1) confer higher exponential growth rates on galactose than any other sugar tested. This hexose transport profile is indicative of the potential for L-arabinose transport, since the galactose transporter 5*. cerevisiae GAL2 is one of the few transporters able to facilitate L-arabinose (45). This correlation is likely due to the similar stereochemistry between L-arabinose and galactose.

[0154] As discovered herein, substitution at the -XXX- positions of the transporter motif sequence uncovered several interesting phenotypes. Indeed, substitution with Thr and Pro (e.g. a transporter motif sequence of -G-G-T38P39T40-G-) results in selective galactose uptake in the modified transporter protein. Such exclusive uptake, as discussed herein, is also indicative of L-arabinose uptake ability (Figures 15 & 16). Thus, the work described herein shows transporter proteins can successfully be engineered into galactose and arabinose transporters.

[0155] This work describes a conserved G-G/F-XXXG motif and an engineering approach to modify this motif. This motif allowed for the rewiring of several transporters and yielded the mutant transporters C. intermedia gxs l Phe38 He39 Met40, S. stipitis rgt2 Phe38 and Met40, and S. cerevisiae hxt7 Ile39Met40Met340 that do not transport glucose yet support S. cerevisiae EX.12 growth on xylose. This motif also yielded C. intermedia Thr28Pro39Thr40 that supports S.

cerevisiae EX.12 growth on galactose, and no other sugar tested. These major facilitator superfamily transporters are channels and thus a substrate molecule interacts with many residues during transport. Yet, no other residues discovered to date display the degree to which glucose transport can be attenuated and xylose transport amplified than the residues in the G-G/F-XXXG motif. Thus, this study provides further insight into the residues responsible for monosaccharide transport in MFS proteins while establishing a platform for engineering a specific, efficient xylose transporter.

[0156] Materials and Methods

[0157] Strains, media, and plasmids - Molecular cloning and standard culturing techniques with E. coli DH10B were performed according to Sambrook (46). S. cerevisiae EX.12 was used for all yeast experiments and was constructed as previously described (38). All transporters were cloned into p414-TEF, a standard yeast shuttle vector created by Mumberg (47). Yeast synthetic complete media was used for culture and experimental growth media. CSM-Trp was used when S. cerevisiae EX.12 was carrying a transporter. Carbon sources were provided at 20 g/L.

[0158] Transporter Cloning - Potential xylose transporters were identified from literature and BLAST search. To obtain this list of 46, we combined 26 transporters from our previous survey of transporters (36) along with 20 additional transporters identified through homology search using C. intermedia GXS1 and S. cerevisiae STL1 as a template. Details on cloning and transporter libraries are described herein. Primers are listed in Table 4 (cloning), Table 5 (saturation mutagenesis), and Table 6 (point mutations).

[0159] Table 4: primers used for cloning putative transporters.

[0160] Table 5: primers used for saturation mutagenesis of C. intermedia GXSl.

[0161] Table 6: Primers used for point mutations.


[0162] Growth rate measurements - All exponential growth rates were measured and calculated according to the method previously described using a Bioscreen C (Growth Curves USA, Piscataway, New Jersey) and a MATLAB script (36, 38).

[0163] Fractional change - Fractional change in growth rate from wild type was calculated by taking the difference between the growth rates of the mutant and wild type over the growth rate of the wild type for each individual carbon source. Error was propagated using the least squares method based on the standard deviation in exponential growth rates of the mutant and the wild type.

[0164] High cell density fermentation - High cell density experiments were conducted as previously described (38). Yeast cultures were suspended at OD in 20 g/L glucose, 10 g/L glucose and 10 g/L xylose, or 20 g/L xylose. Supernatant concentration of xylose and/or glucose was measured using a YSI Life Sciences Bioanalyzer 7100MBS.

[0165] Radiolabeled xylose uptake - Uptake of 14C labeled xylose was used to determine the Michaelis-Menten parameters for C. intermedia GXS1 and the Phe38 He39 Met40 triple mutant. The method was performed as previously described (38).

[0166] Growth rate measurements - All exponential growth rates were measured and calculated according to the method previously described using a Bioscreen C (Growth Curves USA, Piscataway, New Jersey) and a MATLAB script. The Bioscreen C measures online optical density for easy and accurate measurement of the growth curves of up to 200 strains at one time. Error was calculated based on biological triplicate in all cases. In all cases, the Bioscreen C was set to maintain a temperature of 30 °C, employ high continuous shaking, and to measure optical density every 10 minutes. A single carbon source per well was used in all experiments save one. Growth on xylose in the presence of increasing concentrations of glucose was measured for C. intermedia gxs l Phe38 Ile39 Met40.

[0167] It is important to note that the environment of the Bioscreen C does not support cultures reaching high optical density and observed values are below OD6oo of 2. This does not reflect the optical densities reached in flasks, which typically approach OD6oo of 10.

[0168] Transporter Cloning - Each of these transporters was functionally analyzed for conferred growth rate on xylose and glucose in 5*. c. EX.12. Genomic DNA and PCR were performed as previously described (36). Using this approach, open reading frames from

Scheffersomyces stipits, Debaryomyces hansenii, Yarrowia lipolytica, and Saccharomyces cerevisiae were cloned using primers listed in Table 4. Mutant transporters and saturation library construction is described below and Primers are listed in Table 5 (saturation) and Table 6 (point).

[0169] Saturation mutagenesis and point mutation - The Strategene Multi mutagenesis kit was used to generate saturation mutagenesis libraries at positions 38, 39, and 40 in C.i. GXSl . Each codon was replaced with the degenerate NNK sequence recommended for use when creating

saturation mutagenesis libraries. It is important to note that the wild type codon was represented in the NNK library for both Val38 and Leu39 thus alternative 3 primers that did not contain the wild type sequence were designed. This subsequently necessitated the design of specific point mutation primers to access certain residues and the use of the Stratagene Quikchange kit. Some single point mutation primers were ordered to complete the saturation libraries. The Stratagene Quikchange mutagenesis kit was used to generate all rational single, double, and triple mutants. Primers are listed in Table 5 (saturation) and Table 6 (point).

[0170] Example 2: Sequence alignment of 54 sequences from major facilitator superfamily sugar transporter proteins. The transporter motif sequence is shown as bolded residues and corresponds as described herein to residue positions 36-41 of C. intermedia GXSl protein.

Dh2C02530p KF'RNF^DKTPNIYNVF'VIASISCISGLMFGIDISSMSLFIGDD YI YFHK- Dh2E01166p KLRLF^DKLPNIY IYVIAJIISCISGLMFGIDISSMSAFLSNDAYLKYFGT- 63

Dh2E01298p KFR FLDKFPNIH VYIVVGISCIS IDIΞSMSLFIGDDKYLDYE'K'S 63

SsHGT2 KFRTF^DRLPNIY VYIIASISCISGiMFGFDISSMSAFIGEDDYKKFFKM 63

Dh2A14300p SLNKBLDKFHTTYNIYVIAMITTISGMHFOFDVSSISAPISBPSYRRFFKY 61

Y10B06391p QVGALQHRFPKLHHPYLTAAVATMGOIiIiFOFDISSVSAFVDTKPYKBYFGY 59

Y10B01342p tnf VHHPYLTAAVATMG3MIiFOFDISSVSAFVGBBKYMKYFGH 43

BmHGT2 MGRIT PY ^LTALiiCTGGjLFGF'DISSMSAI I SSPNYLTYE'GPKDLTVECPD 52

At5g£92S0 LASDAPBSFSWSSVILPFIPP-ALGOIiIiFSYDIGATSGATLSLQSP ALSGTTWFNF 139

At5gl7010 HVPENYSWAAILPFLFP-ALGqi^Y^BIGATSCaTISIgBPMTLLSYYAVPFSAV 89

SsAUTl LKAEAJI KWHIPPRLIGVIALGS AAAVQGMDESVING/i LFYPKA TMHNSD 161

Y10D00132 LKREIT KWDHPM VYYL ^CGSLj^VQGMDETVING7A II PAQFGI EDSGVVSRKS 180

BmSTLl E1LGM GIKLNWAIGE1AASAGFLLFGYDQGVLGSLYTLPSMNAQETEItJTAAVGDS 73

S3XUT6 A TNSYLGLRGHKLNFAVSCEAGVGFLLFgYDQGVMGSLLTLPSFEKTFPAiyi 75

Dh2E01386p - -KTtJTMGLRGKPLRVAITICCTIGFSLFGYDQGLMSGI ITGKQFlSfEEE'PPTHGT 59

Dh2B05060p - -RTKT SIiRGKRI^V¾FTVVATL8F8IiFSYDQGLWSGLITGEQF ABFPPTAGK 60

SsSTLl - -RTiCTFGIiRGKKLRAFITVVAVTOFSIiFSYDQGLWSGIITADQF SBFPATR 60

ScSTLl - -RTSHWGLTGKKLRYFITIASMTOFSIiFSYDQGLWASLITGKQF YBFPATKBNG D 70

BmHXTIO IDVGLRG WLLTVITA8CAAGFLLVGYDKGVMGGVVGLGEF KTFKNPD 66

SsXUT2 GKgVSYAVTFTCEIAFILFOIBQGIIGNLINNQDFLRTFGNPTG 53

CnBC3990p - -HKlORRLVGHKI^YSVSVFLSI6yi¾F-¾DQGVMSGIITGPYFKftYFKQPTS 62

Y"10F06776p MFSLTGKPLLYFTSVFVSLGVFIJFGYDQGVMSGI ITGFYFKEYFHEPTR 49

BrnXUT3 VGATGAKGLIKNARTFAIAVFASMGGLIYGYNQGMFGQIL3MHSFQEASGVKGIT 78

SsXUTl AGKSGVAGLVANSRSFFIAVFASLOgLVYgY QGMFGQISG YSFSKAIGVEKIQD 77

SsXUT3 AHGNVVT MKIiPVVFLVILFASLGGLLFgYDQGVISG VTMESF- -GAKFPRI M 63

SSXDT3-A AHGNVVT MKIiPVVFLVILFASLGGLLFgYDQGVISG VTMESF- -GAKFPRI M 63

SSXDT3-B AHGNWTIMMKDP FLVILFASLGGLLFGYDQGyiSGIVTMESF- -GAKFPRI M 63

DhXylHP SKGNI ITVMSKDPLVFCI IAFASIGGLLFGYDQGVISGIVTMESF- -AAKFPRI S 64

ScGAL2 PIEI PKKP SEYVTVSLLCLCVAFGGFMFGWDTGTISGFVVQTDFLRRFG-MKHKDGT- - 113

ScHXTS EVWPEKPASAYATVSTMCLCMAFGGFMSQ¾DTGTISGFVNQTDFLRRFGNYSHSKNT-- 109

ScHXTl AVAPPNTGKGVYV v'S ICCVMVAFGGFIFGWDTGTISGFVAQTDFLRRFG-MKIIHDG3 - - 107

ScHXT3 VLTNPNTGKGAYVTVSICCV VAFGGF¥FQ¾DTGTISGFVAQTDFLRRFG-¾KIiKDGS-- 104

SCHXT7 VVEIPKRPASAYVTVSTMCI IAFGGF¥FQ¾DTGTISGFINQTDFIRRFG-¾KIiKDGT-- 107

ScHXT9 PIDLPQKPLSAYTTVAILCL IAFGGFIFQ¾DTGTISGFVNLSDFIRRFG-QK DKGT-- 103

ScHXT2 NAELPAKPIAAYW v'ICLCLMIAFGGFVFGWDTGTISGFV QTDFKRRFG-QMKSDGT- - 98

ScHXTIO SLDI PYKPI IAYWTVMGLCL IAFGGFIFGWDTGTISGFINQTDFKRRFG- ELQRDGS - - 91

CiGXFl QVDAPQKGFKDYIVISIFCF VAFGGFVFQFDTGTISGFVNMSDFKDRFG-QHHADGT-- 86

ScHXT13 VEPPKRGLIGYL IYLLCYPI SFGGFLPGWDSGITAGFINMDb7FKMMFGSYKII5TGE- - 100

BrnGXFl -MVFQVRGTPIGALTLFIAMLAS GGFLFQ¾DTGQISGLTQMADFRQRFATVDNPDAIG- 58

SCHXT14 GQAAKI3HNASLHIPVLLCLVI SLGGFIFGWDIGTIGGMTNMVSFQEKFGTTNI IHDDET 105

BrnGXSl GPVARPASVKQSLPAILVAAASAFGGVLFQYDTGTISGLIVMPKFQETFGKPVPGSTTGA 74

BrnRGT2 GPVARPASVKQSLPAILVAAASAFGGVLFGYDTGTISGIJIVMPNFQETFGKPVPGSTTGA 74

CiGXSl FV VGEKKAGSTAMAI IVGLFAA5GGVLFGYDTGTISGV TMDYVLARY PSNK- 64

CiGXSl-A FV VGEKKAGSTAMAI IVGLFAA5GGVLVGYDTGTISGV TMDYVLARY PSNK- 64

CiGXSl-B FV VGEKKAGSTAMAI IVGLFAAFGGVLSGYDTGTISGV TMDYVLARY PSNK- 64

Dh2DG1474 YV VGEKRAGSASM I VGAFAAFGGVLFGYDTGTISGIMAMNYVKGEF PANK- 64

DhOD02167p YV VGEKRAGSASMGI Fv'GAFAAFGGVLFGYDTGTISGIMAMNYVKGEF PANK- 64

SsRGT2 YINFGEKKAGSTTMGICVGLFAAFGGILFQYDTGTISGI AMDYVTARF PSNH- 64

Y10CG6424p IINRGEKPEGSAFMAAFVAVFVAFGGILFQYDTGTISGV AMPFVKKTF TDDG- 58

Y10C08943p MAI IVAVFVAFGGLLYGYDTGTIAGIMTMGYVKEKF TDFGK 41

D 2B14278p YYKKMQQKS -SS5SAITVGLVAAVGGFLYGYDTGLIK!DIMEMTYVKDNF PANG- 69

Ec.XylE M TQY SSY S LVA LGGLLFGYDTAVISGTVESLH VFVAPONLSESAA - 54

Ss UTB RSIGPLIPR KHLFYGSVLLMS IVHPTIMGYDSMMVGSILNLDAYV YFH 53

ScMALll KSMTLKQALLKYPKAALWSILVSTTLVMEGYDTALLSAIJYAIJPVFQRKFG L GEGS■■ 143

[0171] Example 3: Sequence alignment of 57 sequences from major faciltator superfamily sugar transporter proteins. Bolded residues correspond to the alignment of conserved residue corresponding to 297 of C. intermedia GXS1 protein.

Dh2C02530p WA.QAKQQj TGMMTLMYYIVYVFOMAGYEG BANLVAS IQYCLKTGMTIPALYFMDKLGR 340

Dh2E01166p FAQIWQQ3 TGMMTLMYYIVYVFEMAGYHG BANLVAS IQYCINFAM IPALYLMDKVGR 340

Dh2E01298p FAQIWQQ3 TGMMTLMYYIVYVFDMAGYQG DANLIAS IQYVLFFVMTAPSLYLMDKLGR 340

SSHGT2 FAQI QQJ TGMMVMMYYIVYIFNMAGYSN NANLVASSIQYVLNTAATVPALFLMDYIGR 340

Dh2A14300p SAQIK QJ TGMNVMMYYIVYIFE VGYTG !\FTVLVSSSIQYVIKFGVTLIALPLSDYVGR 336

Y10B06391p WAQI QQJ GMMVMMYYIVLIFTMAGYTG NANLVASSIQYVIKMIMTIPALLFIDRVGR 336

Y10B01342p WAQI QQJ LTGMMIMMYYWI IFKMAGYSGKSAVIVSGSIQYI INWMTIPALLFIDKIGR 320

BmHGT2 E'TQI WSQL GIMVM Y LSYVFEMAGITG- IALISNGIQYVIIWVMTCPALLYVBRWGR 347

At5g£92S0 GLVLFQQITGQPSVLY YAGSILOTAGFSAAADATRVSVIIGVFKLLMTWVAVAKVDDLGR 424

At5gl7010 GLVL QQ LIMTGVAVVVIDRLGR 334

SsAUTl E'LVMFMQQE'CGILWIAYYSSSI P^QSGFSQTSALIASWGE'GMLNFTFAI AE'FTIBRFGR 441

Y10D00132 FIVMFMQQFCGIOTIAYYSSNIP¾ESGFGAIQALIJISFGFGAINF\'FALPAVYTIBTFGR 459

BrnSTLl SQMFQQISGINLITYYIGKTLQEQLGFSBINSRILAAANGTEYFIASWAAVFFIEKMGR 353

S3XUT6 WSQIMQQI GINIITYYAGTI ESYIGMSPF SRILAALNGTEYFLVSLIAFYTVERLGR 363

Dh2E01386p STOFFQQFTGC ASIYYSTVLFE SIGLTG LPLILGGVFATIYALSTIPSFFLIBRLGR 344

Dh2B05060p SGOFFOQFTGCNAAIYYSTVLFEDTIHLERRLALILGGVFATVYALSTIPSFFLVDTLGR 345

SsSTLl STQ 'FQQFTGCNAAIYYSTVLFODTIGLERRMALIIGGVFATVYAIFTIPSFFLVDTLGR 342

ScSTLl STQE'FQQFTGCNAAIYYSTVLFNKTIKLDYRLSMIIGGVFATIYALSTIGSFFLIEKLGR 356

BmHXTIO FIQAAQQLSGINALI YSGTLFSQSIGLDSKKSALFAGGLNMCLILGSTISIFLIDRVGR 346

SsXUT2 SMFAQQLSGVNVVNYYITE'VLI SVGIED LALILGGVAVICFTVGSLVPTFE'ABR GR 330

CnBC3990p SSOLFAQLNGINVISYTAPLVFEQAG-W GRBAILMTGINALFYVASSLPPWYLMBRAGR 334

Y10F06776p SSOMFAQLNGINVISYTAPLVFEEAG-WGRSAILMTGINGIVYVCST PPWYLVBKWGR 322

Dh2F19140p FSOMFAQLNGINMVSYYAPKFELΛG-KVGRQAILMTGI 8IVY^LSTIP ¾YLVBG¾GR 293

SsXUT4 E'SO FAQ NG^NMVSYYAPMIFE3J\G-WVGRQAIL TGIN3IIY'I ^ IPPWYL¾RBSWGR 293

SsXUT7 SALGFAQFNGINIIS TAPMVFEEAG-FNNSKALLMTGINSIVYWFSTIPPWFLVBHWGR 274

BrnXUT3 LIMLFOQWTGI FILYYAPFIFKQIGLSGKTISLLASGVVGIVLFLATIPAVLYIBSWGR 382

SsXUTl LIMTFQQWTGV FILY YAPFIFSSLGLSGNTISLLASGWGIVMFLATIPAVLKVDRLGR 381

SsXUT3 A MFFOQFIGCNAIIY YAPTIFTQLGMNSTTTSLLGTGLYGIVNCLSTLPAVFLIDRCGR 381

SsXUT3-A AVMFFOQFIGCNAIIY YAPTIFTQLGMNSTTTSLLGTGLYGIVNCLSTLPAVFLIDRCGR 381

S3XUT3-B AVMFFOQFIGCNAIIY YAPTILTRLGMNSTTTSLLGTGLYGIVNCLSTLPAVFLIDRCGR 381

DhXylHP AVMFFOQFIGCNAIIYYAPTIFSQLGMDSNTTALLGTGVYGIVNCLSTIPAIFAIDRE'GR 382

ScGAL2 FVOMFOQLTG NYFFYYGTVIFK3VGLDD SFETSIVIGVVNFASTFFSLKTVENLGH 392

ScHXTS MIN3LOQLTGBNYFFYYGTTIFK3VGMND- SFETSIVLGIVNFASCFFSLYSVDKLGR 388

ScHXTl MI03LOQLTGBNYFFYYGTIVFOAVGLSD- SFETSIVFGWNFFSTCCSLYTVBRFGR 386

ScHXT3 IQSLQQLTGDNYFE'Y'YGTTVFNAVGMSD- SFETSIVFGWNFFSTCCSLYTVBRFGR 383

ScHXT7 IQSLQQLTGDNYFE'Y'YGTTI KAVGLSD- SFETΞIVLGIVNFA3TFVGI YVVERYGR 386

ScHXT9 MI03LOQLTGBNYFFYYGTTIFK3VGLKD- SFQTSIIIGVVNFFSSΕΊAVYTIERE'GR 382

ScHXT2 IQSLQQLTGNNYFE'Y'YGTTI NAVGMKB- SFQTSIVLGIVNFASTFVALYTVBKFGR 377

ScHXTIO VIQSLQQLTGCNYFFYYGTTIFNAVGMQD- S ETSIVLGAVNFASTFVALYIVBKFGR 370

CiGXFl MLQ3LQQLTGDNYFFYYGTTIFQAVGLKD- S QTSI ILGIVNFASTFVGIYVIERLGR 365

ScHXT13 LVQTFLQLTGENYFFFYGTTIFKSVGLTD- GFETSIVLGTVNFE'STI IAVMVVDKIGR 379

BrnGXFl TLQAGQQFTGANYFFYFGTAIFTSVGLSD- 3FVTQI ILGAVNFACTFLGLΥΊLERFGR 340

SCHXT14 MIMAFQQLSGINYFFYYGTSVFKGVGIKD- PYITS11L3SVNFLSTILGIYYVEKWGH 403

BrnG Sl FIQAFQQLTGINFIFY GTKFFKSALPGTN PFIFSVISNVVNVVTTVPGMYM ERLGR 354

BrnRGT2 FIQAFQQLTGINFIFYYGTKFFKSALPGTN PFIFSVISNVVNVVTTVPGMYMMERLGR 730

CiGXSl AIQAFQQLTGV FIFYYGTTFFKRAG vTsl - - GFTISLATNIVNVGSTIPGILLMEVLGR 342

CiGXSl-A AIQAFQQLTGV FIFYYGTTFFKRAG vTsl - - GFTISLATNIVNVGSTIPGILLMEVLGR 342

CiGXSl-B AIQAFQQLTGV FIFYYGTTFFKRAG vTsl - - GFTISLATNIVMVGSTIPGILLMEVLGR 342

Dh2DG1474 ALQAFQQLTGV FIFYFGTSFFKSAGIEN- EFLISLAT3IVNVGMTVPGIFLIELVGR 343

DhOD02167p ALQAFQQLTGV FIFYFGTSFFKSAGIEN- EFLISLAT31VSVGMTVPGI FLIELVGR 343

SsRGT2 GIQALQQLTGINFIFYYGTNFFKGSGIKN- EFLIQMATNIVNFGSTVPGILLVE11GR 343

Y10CG6424p AIQALQQLTGINFIFYYGTEFFKKSNISN- PFLIQMITNIVNVVMTIPGIMFVBRVGR 336

Y10C08943p SIQALQQLTGINFIFYYGTNFFKTAGIKD- PFVVSMITSAVNVAFTLPGILFVDKVGR 319

Dh2B14278p GVQAFQQSSGINFIFYYGVKFFASSGIKK- YYLMSFVTYAVNTLFTIPGIILIEVIGR 351

EcXylE MLSIFQQFVGINVVLYYAPEVFKTLGAST-DIALLQTIIVGVINLTFTVLAIMTVBKFGR 341

SsXUTS TQAIVTEMAGSSVGSYYFSIILTQAGVKDSNDRLRV IVMSSWSLVIALSGCLMFDRIGR 331

ScMALll CLT VAQNSSGAVLLGYSTYFFERAGMAT-DKAFTFSLIQYCLGLAGTLCSWVISGRVGR 431

[0172] References

1. Reijenga KA, et al. (2001). Biophysical Journal 80(2):626-634.

2. Gardonyi M, Jeppsson M, Liden G, Gorwa-Grausland MF, & Hahn-Hagerdal B (2003). Biotechnology and Bioengineering 82(7):818-824.

3. Elbing K, et al. (2004). Applied and Environmental Microbiology 70(9):5323-5330.

4. Wahlbom CF, Otero RRC, van Zyl WH, Hahn-Hagerdal B, & Jonsson LJ (2003). Applied and Environmental Microbiology 69(2):740-746.

5. Bengtsson O, et al. (2008). Yeast 25(11):835-847.

6. Jeffries TW & Jin YS (2004). Applied Microbiology and Biotechnology 63(5):495-509. 7. Hahn-Hagerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, & Gorwa-Grauslund

MF (2007). Applied Microbiology and Biotechnology 74(5):937-953.

8. Martin CH, Nielsen DR, Solomon KV, & Prather KLJ (2009). Chemistry & Biology 16(3):277-286.

9. Tyo KEJ, Kocharin K, & Nielsen J (2010). Current Opinion in Microbiology

13(3):255-262.

10. Curran KA & Alper HS (2012). Metabolic Engineering 14(4):289-297.

11. Hahn-Hagerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, & Zacchi G (2006). Trends in Biotechnology 24(12):549-556.

12. Almeida JR et al. (2007). Journal of Chemical Technology & Biotechnology

82(4):340-349.

13. Van Vleet JH & Jeffries TW (2009). Curr Opin Biotechnol 20(3):300-306.

14. Zhang F, Rodriguez S, & Keasling JD (2011). Current Opinion in Biotechnology 22(6):775-783.

15. Liu L, Redden H, & Alper HS (2013). Current Opinion in Biotechnology. DOI:

10.1016/j.copbio.2013.03.005.

16. Hong KK & Nielsen J (2012). Cellular and molecular life sciences : CMLS

69(16):2671-2690.

17. Bae JY, Laplaza J, & Jeffries TW (2008). Applied Biochemistry and Biotechnology 145(l-3):69-78.

18. Karhumaa K, Pahlman AK, Hahn-Hagerdal B, Levander F, & Gorwa-Grauslund MF (2009). 7eosi 26(7):371-382.

19. Runquist D, Hahn-Hagerdal B, & Bettiga M (2010). Applied and Environmental Microbiology 76(23):7796-7802.

20. Krahulec S, Klimacek M, & Nidetzky B (2012). Journal of Biotechnology 158(4): 192-202.

21. Lee SM, Jellison T, & Alper HS (2012). Applied and Environmental Microbiology 78(16):5708-5716.

22. Scalcinati G, et al. (2012). FEMS Yeast Res 12(5):582-597.

23. Jojima T, Omumasaba CA, Inui M, & Yukawa H (2010). Applied Microbiology and Biotechnology 85(3):471-480.

24. Young E, Lee SM, & Alper H (2010). Biotechnology for Biofuels 3(24):24.

25. Boles E & Hollenberg CP (1997). FEMS Microbiology Reviews 21(1):85-111.

26. Wieczorke R et al. (1999). FEBS Letters 464(3): 123-128.

27. Pao SS, Paulsen IT, & Saier MH, Jr. (1998). Microbiology and Molecular Biology Reviews 62(1): 1-34.

28. Ozcan S & Johnston M (1999). Microbiology and Molecular Biology Reviews

63(3):554.

29. Sedlak M & Ho NWY (2004) Char. Yeast 21(8):671-684.

30. Subtil T & Boles E (2012). Biotechnology for Biofuels 5: 14.

31. Leandro MJ, Goncalves P, & Spencer-Martins I (2006). Biochemical Journal 395:543-549.

32. Saloheimo A, et al. (2007). Applied Microbiology and Biotechnology 74(5): 1041-1052.

33. Hector RE, Qureshi N, Hughes SR, & Cotta MA (2008). Applied Microbiology and Biotechnology 80(4):675-684.

34. Katahira S, et al. (2008). Enzyme and Microbial Technology 43(2): 115-119.

35. Du J, Li SJ, & Zhao HM (2010). Molecular Biosystems 6(11):2150-2156.

36. Young E, Poucher A, Comer A, Bailey A, & Alper H (2011). Applied and

Environmental Microbiology 77(10):3311-3319.

37. Leandro MJ, Fonseca C, & Goncalves P (2009). FEMS Yeast Research 9(4):511-525. 38. Young EM, Comer AD, Huang HS, & Alper HS (2012) A. Metabolic Engineering 14(4):401-411.

39. Kasahara T & Kasahara M (2010). Journal of Biological Chemistry 285(34):26263-26268.

40. Ha SJ, et al. (2013). Applied and Environmental Microbiology 79(5): 1500-1507. 41. Sun L, et al. (2012) Cry. Nature 490(7420):361-366.

42. Hamacher T, Becker J, Gardonyi M, Hahn-Hagerdal B, & Boles E (2002).

Microbiology 148(Pt 9):2783-2788.

43. Kasahara T, Shimogawara K, & Kasahara M (2011). Biochemistry 50(40):8674-8681.

44. Davis EO & Henderson PJF (1987). Journal of Biological Chemistry 262(29): 13928-13932.

45. Subtil T & Boles E (2011). Biotechnology for Biofuels 4:38.

46. Sambrook J (2000) Molecular Cloning: A Laboratory Manual, ed Russell DW (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

47. Mumberg D, Muller R, & Funk M (1995). Gene 156(1): 119-122.