WO2013155481A1

WO2013155481A1 - Improved cellodextrin transport and mixed sugar fermentation

Info

Publication number: WO2013155481A1
Application number: PCT/US2013/036480
Authority: WO
Inventors: James H. Doudna Cate; Jonathan M. Galazka; Yong-Su Jin; Suk-Jin Ha
Original assignee: The Board Of Trustees Of The University Of Illinois; The Regents Of The University Of California
Priority date: 2012-04-13
Filing date: 2013-04-12
Publication date: 2013-10-17

Abstract

The present disclosure relates to compositions and methods for increasing the transport of cellodextrin into cells, for increasing growth of cells, for increasing synthesis of hydrocarbons and hydrocarbon derivatives, and for co-fermenting cellulose-derived and hemicellulose-derived sugars.

Description

IMPROVED CELLODEXTRIN TRANSPORT AND MIXED SUGAR FERMENTATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/624,235, filed April 13, 2012, which is hereby incorporated by reference, in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 658012001340SEQLIST.TXT, date recorded: April 11, 2013, size: 74 KB)

FIELD

[0003] The present disclosure relates to compositions and methods for increasing the transport of cellodextrin into cells, for increasing growth of cells, for increasing synthesis of hydrocarbons and hydrocarbon derivatives, and for co-fermenting cellulose-derived and hemicellulose-derived sugars.

BACKGROUND

[0004] Biofuels are under intensive investigation due to the increasing concerns about energy security, sustainability, and global climate change (Lynd et al., 1991). Bioconversion of plant- derived lignocellulosic materials into biofuels has been regarded as an attractive alternative to chemical production of fossil fuels (Lynd et al. 2008; Hahn-Hagerdal et al. 2006).

Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin.

[0005] The engineering of microorganisms to perform the conversion of lignocellulosic biomass to ethanol efficiently remains a major goal of the biofuels field. Much research has been focused on genetically manipulating microorganisms that naturally ferment simple sugars to alcohol to express cellulases and other enzymes that would allow them to degrade

lignocellulosic biomass polymers and generate ethanol within one cell. However, an area that has been less well studied is that of sugar transporters. An understanding of the regulation of sugar transport and the genetic engineering of microorganisms to have improved sugar-uptake ability will greatly improve efficiency (Stephanopoulos 2007). Furthermore, other types of proteins involved in the regulation of cellulase expression and activity remain to be fully explored.

[0006] Saccharomyces cerevisiae, also known as baker's yeast, has been used for bioconversion of hexose sugars into ethanol for thousands of years. It is also the most widely used microorganism for large scale industrial fermentation of D-glucose into ethanol. S.

cerevisiae is a very suitable candidate for bioconversion of lignocellulosic biomass into biofuels (van Maris et al, 2006). It has a well-studied genetic and physiological background, ample genetic tools, and high tolerance to high ethanol concentration and inhibitors presented in lignocellulosic hydrolysates (Jeffries 2006). The low fermentation pH of S. cerevisiae can also prevent bacterial contamination during fermentation.

[0007] Unfortunately, wild type S. cerevisiae cannot utilize pentose sugars (Hector et al., 2008). To overcome this limitation, pentose utilization pathways from pentose-assimilating organisms have been introduced into S. cerevisiae, allowing fermentation of D-xylose and L- arabinose (Hahn-Hagerdal et al, 2007; Brat et al, 2009; Wisselink et al, 2007, 2009;

Wiedemann and Boles 2008; Karhumma et al, 2006). However, efficient conversion of pentose sugars into biofuels is limited by multiple issues including cellular redox imbalance, low influx of pentose phosphate pathway, and lack of efficient pentose transport into the cell (Hector et al, 2008).

[0008] In addition, both natural and engineered microorganisms show reduced ethanol tolerance during xylose fermentation as compared to glucose fermentation (Jeffries and Jin 2000). Combined with the lower fermentation rate, the reduced ethanol tolerance during xylose fermentation poses a significant problem in fermentation of sugar mixtures containing the high concentrations of glucose (~ 70-100 g/L) and xylose (~ 40-60 g/L) present in cellulosic hydrolysates. Since microorganisms utilize glucose preferentially, at the time of glucose depletion (when cells begin to use xylose), the ethanol concentration is already high enough (~ 35-45 g/L) to further reduce the xylose fermentation rate. As a result, sequential utilization of xylose after glucose depletion because of "glucose repression" is a significant challenge to be overcome in order to successfully utilize mixed sugars in cellulosic hydrolysates.

[0009] One solution to this problem is to use cellodextrins, such as cellobiose, rather than glucose. U.S. Patent Application Publication No. US 2011/0020910 discloses that cellobiose (a disaccharide of glucose) can be used as a carbon source in a host cell instead of glucose in order to remove the inhibition on xylose utilization by glucose. This was achieved by increasing the transport of cellodextrin into a host cell by expressing a Neurospora crassa cellodextrin transporter in the cell.

[0010] However, while U.S. Patent Application Publication No. US 2011/0020910 discloses Neurospora crassa genes, and homologs thereof, that encode transporter proteins that increase cellodextrin transport into cells, there still exists a need for the identification and development of modified genes that improve the transport of sugars such as cellodextrin, as well as improve the degradation of lignocellulose, and for their use in the engineering of microorganisms for superior growth on lignocellulose and uptake of compounds resulting from lignocellulose degradation. A further need exists for improved methods of efficient conversion of pentose sugars into biofuels and of mixed sugar fermentation for the production of biofuels.

BRIEF SUMMARY

[0011] In order to meet the above needs, the present disclosure provides novel host cells containing recombinant polynucleotides encoding mutant cellodextrin transporters that increase the rate of cellodextrin transport into the cell, and methods of using such host cells for increasing transport of cellodextrin into a host cell, increasing growth of a host cell on a medium containing cellodextrin, co-fermenting cellulose-derived and hemicellulose-derived sugars, and making hydrocarbons or hydrocarbon derivatives. The mutant cellodextrin transporters of the present disclosure each have a V_», that is at least 1-fold higher than the V_», of a corresponding non- mutant cellodextrin transporter, and a K_m that is at least 0.2-fold higher than the K_m of a corresponding non-mutant cellodextrin transporter. Advantageously, the mutant cellodextrin transporters of the present disclosure result in increased cellodextrin transport into a host cell, which allows the cell to utilize cellodextrin rather than glucose, thus removing the inhibition on xylose utilization. Moreover, the mutant cellodextrin transporters of the present disclosure have improved kinetic properties that result in host cells that are able to more efficiently convert cellodextrins, such as cellobiose, to fermentation products such as ethanol or butanol.

[0012] Accordingly, certain aspects of the present disclosure provide a host cell containing, a recombinant polynucleotide encoding a mutant cellodextrin transporter, where the mutant cellodextrin transporter contains a transmembrane a-helix 1, a-helix 2, a-helix 3, a-helix 4, a- helix 5, a-helix 6, a-helix 7, a-helix 8, a-helix 9, a-helix 10, a-helix 11, a-helix 12, and a loop sequence positioned between a-helix 6 and a-helix 7, where the mutant cellodextrin transporter contains at least one mutation in the loop sequence, and where the mutant cellodextrin transporter has a V„ that is at least 1-fold higher than the V_», of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence.

[0013] In certain embodiments, the at least one mutation is an amino acid substitution. In certain embodiments that may be combined with any of the preceding embodiments, the loop sequence corresponds to amino acids 237-306 of SEQ ID NO: 1. In certain embodiments, the at least one mutation is an amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments, the at least one mutation is selected from an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Cys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Phe amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Lys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Leu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Asn amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Thr amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, and an Ala to Val amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments, the at least one mutation is an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments, the at least one mutation is an Ala to Arg amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments, the at least one mutation is an Ala to Lys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments, the at least one mutation is an Ala to Glu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments, the at least one mutation is an Ala to Gin amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1. In certain embodiments that may be combined with any of the preceding embodiments,

transmembrane a-helix 1 contains SEQ ID NO: 3. In certain embodiments that may be combined with any of the preceding embodiments, transmembrane a-helix 2 contains SEQ ID NO: 4. In certain embodiments that may be combined with any of the preceding embodiments, the mutant cellodextrin transporter further contains a loop connecting transmembrane a-helix 2 and transmembrane a-helix 3 containing SEQ ID NO: 5. In certain embodiments that may be combined with any of the preceding embodiments, transmembrane a-helix 5 contains SEQ ID NO: 6. In certain embodiments that may be combined with any of the preceding embodiments, transmembrane a-helix 6 contains SEQ ID NO: 7. In certain embodiments that may be combined with any of the preceding embodiments, the mutant cellodextrin transporter further contains a sequence between transmembrane a-helix 6 and transmembrane a-helix 7 containing SEQ ID NO: 8. In certain embodiments that may be combined with any of the preceding embodiments, transmembrane a-helix 7 contains SEQ ID NO: 9. In certain embodiments that may be combined with any of the preceding embodiments, transmembrane a-helix 10, transmembrane a-helix 11, and the sequence between a-helix 10 and a-helix 11 contain SEQ ID NO: 10. In certain embodiments that may be combined with any of the preceding embodiments, the mutant cellodextrin transporter further contains a loop connecting transmembrane a-helix 2 and transmembrane a-helix 3 containing SEQ ID NO: 5, transmembrane a-helix 5 contains SEQ ID NO: 6, transmembrane a-helix 6 contains SEQ ID NO: 7, the mutant cellodextrin transporter further contains a sequence between transmembrane a-helix 6 and transmembrane a-helix 7 containing SEQ ID NO: 8, and transmembrane a-helix 10, transmembrane a-helix 11, and the sequence between a-helix 10 and a-helix 11 contain SEQ ID NO: 10. In certain embodiments that may be combined with any of the preceding embodiments, the mutant cellodextrin transporter contains an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to SEQ ID NO: 2. In certain embodiments that may be combined with any of the preceding embodiments, the V_», of the mutant cellodextrin transporter is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, or at least 6-fold higher than the V_», of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence. In certain embodiments that may be combined with any of the preceding embodiments, the mutant cellodextrin transporter has a K_m that is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold higher than the K_m of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence. In certain embodiments that may be combined with any of the preceding embodiments, the host cell exhibits a cellodextrin consumption rate that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65- fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2- fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, or at least 3-fold higher than the cellodextrin consumption rate exhibited by a corresponding cell lacking the mutant cellodextrin transporter. In certain embodiments that may be combined with any of the preceding

embodiments, the host cell exhibits an ethanol productivity that is at least 0.2-fold, at least 0.25- fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8- fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75- fold, or at least 3-fold higher than the ethanol productivity exhibited by a corresponding cell lacking the mutant cellodextrin transporter. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains a recombinant polynucleotide encoding at least a catalytic domain of a cellodextrin phosphorylase. In certain embodiments, the cellodextrin phosphorylase contains an amino acid sequence that has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. In certain embodiments, the cellodextrin phosphorylase has cellobiose phosphorylase activity. In certain embodiments, the cellodextrin phosphorylase with cellobiose phosphorylase activity contains an amino acid sequence that has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25. In certain embodiments that may be combined with any of the preceding

embodiments, the host cell further contains a recombinant polynucleotide encoding at least a catalytic domain of a β-glucosidase. In certain embodiments, the β-glucosidase is from

Neurospora crassa. In certain embodiments, the β-glucosidase is encoded by NCU00130. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains one or more recombinant polynucleotides encoding one or more enzymes involved in pentose utilization. In certain embodiments, the one or more enzymes are selected from one or more of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase. In certain embodiments that may be combined with any of the preceding embodiments, the host cell further contains a recombinant polynucleotide encoding a pentose transporter. In certain embodiments, the pentose transporter is selected from NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3. In certain

embodiments that may be combined with any of the preceding embodiments, the cellodextrin is selected from one or more of the group consisting of cellobiose, cellotriose, and cellotetraose. In certain embodiments that may be combined with any of the preceding embodiments, the host cell is an oleaginous yeast. In certain embodiments that may be combined with any of the preceding embodiments, the host cell is selected from Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus,

Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Myceliophthora thermophila, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Bacillus subtilis, Rhodosporidium toruloides, Lipomyces starkyei, Yarrowia lipolytica, and Cryptococcus curvatus.

[0014] Other aspects of the present disclosure provide a method of increasing transport of cellodextrin into a cell, by: providing the host cell of any one of the preceding embodiments; and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared to a corresponding cell expressing a recombinant polynucleotide encoding a cellodextrin transporter lacking the at least one mutation in the loop sequence.

[0015] In certain embodiments, the cellodextrin is transported into the cell at a V_», that is at least 1-fold higher than the V_», of cellodextrin transport into a corresponding cell having a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence. In certain embodiments, the cellodextrin is transported into the cell at a V„ that is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, or at least 6-fold higher than the V„ of cellodextrin transport into a corresponding cell having a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence. In certain embodiments that may be combined with any of the preceding embodiments, the cellodextrin is consumed at a rate that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75- fold, or at least 3-fold higher than the rate of cellodextrin consumption by a corresponding cell lacking the mutant cellodextrin transporter. In certain embodiments that may be combined with any of the preceding embodiments, the method further includes culturing the host cell under conditions sufficient to ferment the cellodextrin. In certain embodiments, the fermentation of the cellodextrin results in the production of a fermentation product. In certain embodiments, the fermentation product is a fuel. In certain embodiments, the fuel is ethanol or butanol. In certain embodiments, the fermentation product is a fuel. In certain embodiments, the fuel is ethanol. In certain embodiments, the ethanol is produced with an ethanol productivity that is at least 0.2- fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75- fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, or at least 3-fold higher than the ethanol productivity of ethanol produced by a corresponding cell lacking the mutant cellodextrin transporter. In certain embodiments that may be combined with any of the preceding embodiments, the medium contains a cellulase- containing enzyme mixture from an altered organism, where the cellulase-containing mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism.

[0016] Other aspects of the present disclosure provide an isolated polypeptide containing an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or at least 100% identical to SEQ ID NO: 2, wherein the polypeptide comprises an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 2. Other aspects of the present disclosure provide an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 2. Other aspects of the present disclosure provide an isolated polynucleotide encoding the polypeptide of any of the preceding embodiments. Other aspects of the present disclosure provide an expression vector, containing the isolated polynucleotide of the preceding embodiment, operably linked to a regulatory sequence. Other aspects of the present disclosure provide a host cell containing the expression vector of the preceding embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows a schematic of the genomic regions containing BGL5, EGC2, and HXT2.4 in S. stipitis.

[0018] Figure 2A shows comparisons of cellobiose fermentation profiles of strains CEN- HXT2.4-BGL and CEN-CDT1-BGL. Fermentation experiments were performed in YP medium containing 80 g/L of cellobiose under oxygen-limited conditions. Symbols: OD (O), cellobiose (A ), and ethanol (♦). Figure 2A shows CEN-HXT2.4-BGL. Figure 2B shows CEN-CDT1- BGL. [0019] Figure 3 shows that the specific growth rate of the CEN-HXT2.4-BGL strain was improved through a directed evolutionary approach by serially transferring the strain to YP medium containing 80 g/L of cellobiose

[0020] Figure 4 shows a comparison of cellobiose fermentation performance by the CEN- CDT1-BGL, CEN-HXT2.4-BGL, and evolved CEN-HXT2.4-BGL strains cultured in YP medium containing 80 g/L of cellobiose under oxygen limited conditions. Symbols: CEN-CDTl - BGL (·), CEN-HXT2.4-BGL (■), and evolved CEN-HXT2.4-BGL ( A ). Figure 4A depicts the amount of cellobiose consumed. Figure 4B depicts cell growth. Figure 4C depicts the amount of ethanol produced. Figure 4D depicts cellodextrin accumulation.

[0021] Figure 5 shows the predicted structure of the HXT2.4 polypeptide. The predicted structure shows that HXT2.4 contains 12 transmembrane helices. The location of the A291D amino acid substitution in the HXT2.4 (A291D) mutant is indicated by the arrow.

[0022] Figure 6 shows the prediction of transmembrane helix of HXT2.4 (A291D). There are 12 helixes (S1-S12) and top and bottom numbers in each helix correspond to the first and last residues in the helixes. The arrow indicated the location of A291D.

[0023] Figure 7 shows an amino acid sequence alignment of the HXT2.4 polypeptide (SEQ ID NO: 19) with that of the cellobiose transporter CDT-1 (SEQ ID NO: 18). Sequence motifs are underlined; and the A291D mutation in HXT2.4 and corresponding amino acid residue in CDT- 1 are depicted in bolded and underlined text.

[0024] Figure 8 shows comparisons of cellobiose fermentation profiles from 10 mutant D452-HXT2.4 (A291X)-BGL strains having different amino acid substitutions at position 291. Fermentation experiments were performed in YP medium containing 80 g/L of cellobiose under oxygen-limited conditions. Symbols: cellobiose (■), OD (O), and ethanol (♦).

[0025] Figure 9 shows comparisons of cellobiose consumption rates, ethanol production rates, and maximum cellodextrin accumulation from all mutant D452-HXT2.4 (A291X)-BGL strains that showed improved cellobiose fermentation capabilities as compared to the wild type HXT2.4 transformant (D452-HXT2.4-BGL). Fermentation experiments were performed in YP medium containing 80 g/L of cellobiose under oxygen-limited conditions. [0026] Figure 10 shows comparisons of cellobiose consumption rates, ethanol production rate, and maximum cellodextrin concentration by mutant D452-HXT2.4 (A291X)-BGL strains depending on amino acid replacement at position 291 of HXT2.4. The number in parenthesis corresponds to how many times the particular residue was selected. Symbols: cellobiose consumption rate (^), ethanol production rate (X), and maximum cellodextrin concentration (□)·

[0027] Figure 11 shows comparisons of cellobiose consumption, cell growth, and ethanol production from representative transformants of each amino acid substitution. Fermentation experiments were performed in YP medium containing 80 g/L of cellobiose under oxygen limited-conditions. Symbols: D452-HXT2.4 (A291D)-BGL strain (·), D452-HXT2.4 (A291E)- BGL strain (O), D452-HXT2.4 (A291K)-BGL strain ( T ), D452-HXT2.4 (A291R)-BGL strain (V), D452-HXT2.4 (A291V)-BGL strain (■), D452-HXT2.4 (A291L)-BGL strain (□), and D452-HXT2.4 (wild type)-BGL strain (♦). Figure 11A depicts cellobiose consumption. Figure 1 IB depicts cell growth. Figure 11C depicts ethanol production.

[0028] Figure 12 shows transport kinetics of wild type HXT2.4 and mutant HXT2.4

(A291D) strains. The linear rate of [ H]-cellobiose uptake into yeast strains with wild type HXT2.4 or mutant HXT2.4 (A291D) was determined using various concentrations of cellobiose. Error bars represent the standard error of three replicate measurements at each concentration. Figure 12A depicts the wild type HXT2.4 strain. Figure 12B depicts the mutant HXT2.4 (A291D) strain.

[0029] Figure 13 shows growth assays of the CDT-1 strain, wild type HXT2.4 transformant strain, and mutant HXT2.4 (A291D) transformant strain on cellobiose, cellotriose, and cellotetraose medium. Fermentations were performed in duplicate independently with less than 5% variations and one of representative experiment is shown. Symbols: D452-CDT1-BGL strain (O), D452-HXT2.4-BGL strain (♦), and D452-HXT2.4 (A291D)-BGL strain ( A). Figure 13A depicts growth assays on cellobiose medium. Figure 13B depicts growth assays on cellotriose medium. Figure 13C depicts growth assays on cellotetraose medium.

[0030] Figure 14 shows comparisons of cellobiose fermentation profiles from 19 D452 HXT2.4 (A291X)-BGL mutant strains that grew faster in YSC agar medium containing 20 of cellobiose. Fermentation experiments were performed in YP medium containing 80 g/L of cellobiose under oxygen-limited conditions. Symbols: cellobiose (■), OD (O), and ethanol (♦)

DETAILED DESCRIPTION

Definitions

[0031] Unless defined otherwise, all scientific and technical terms are understood to have the same meaning as commonly used in the art to which they pertain. For the purpose of the present disclosure, the following terms are defined.

[0032] As used herein, "cellodextrin" refers to glucose polymers of varying length and includes, without limitation, cellobiose (2 glucose monomers), cellotriose (3 glucose monomers), cellotetraose (4 glucose monomers), cellopentaose (5 glucose monomers), and cellohexaose (6 glucose monomers).

[0033] As used herein, a "cellodextrin transporter" refers to any sugar transport protein capable of transporting cellodextrins across the cell membrane of a cell.

[0034] As used herein, "sugar" refers to monosaccharides (e.g. , glucose, fructose, galactose, xylose, arabinose), disaccharides (e.g., cellobiose, sucrose, lactose, maltose), and

oligosaccharides (typically containing 3 to 10 component monosaccharides).

[0035] As used herein, "lignocellulose" refers to any material primarily consisting of cellulose, hemicellulose, and lignin.

[0036] The term "hemicellulose" refers to a polymer of short, highly-branched chains of mostly five-carbon pentose sugars (e.g. , xylose and arabinose) and to a lesser extent six-carbon hexose sugars (e.g., galactose, glucose and mannose).

[0037] As used herein, "V^_H" refers to the maximum rate that a cellodextrin transporter polypeptide of the present disclosure transports cellodextrin into a host cell. [0038] As used herein, "K_m" refers to the concentration of a cellodextrin transporter polypeptide of the present disclosure at which the rate that the polypeptide transports

cellodextrin into a host cell is half of ν_^.

[0039] As used herein, "1-fold higher" refers to a 100% increase. For example a V_», of 2 would be 1-fold higher than a V_», of 1.

[0040] As used herein, "0.5-fold higher" refers to a 50% increase. For example a K_m of 60 μΜ would be 0.5-fold higher than a K_m of 40 μΜ.

Overview

[0041] The present disclosure relates to host cells containing a recombinant polynucleotide encoding a mutant cellodextrin transporter containing a transmembrane a-helix 1, a-helix 2, a- helix 3, a-helix 4, a-helix 5, a-helix 6, a-helix 7, a-helix 8, a-helix 9, a-helix 10, a-helix 11, a- helix 12, and a loop sequence positioned between a-helix 6 and a-helix 7, where the mutant transporter contains at least one, at least two, or more mutations in the loop sequence, and where the mutant cellodextrin transporter has a V„ that is at least 1-fold higher than the V„ of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence. As used herein, "1-fold higher" refers to a 100% increase. For example a V„ of 2 would be 1-fold higher than a of 1.

[0042] The present disclosure also relates to using such host cells to, for example, increase cellodextrin transport into a host cell, increase growth of a host cell on a medium containing cellodextrin, co-fermentcellulose-derived and hemicellulose-derived sugars, and make hydrocarbons or hydrocarbon derivatives. Moreover, the present disclosure is based, at least in part, on the discovery, isolation, and production of a novel mutant Scheffersomyces (formerly Pichia) stipitis HXT2.4 cellodextrin transporter containing an alanine (Ala) to aspartate (Asp) amino acid substitution at position 291 that has a V„ that is at least 1-fold higher than the V„ of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence; and a K_m that is at least 0.5-fold higher than the K_m of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence. As used herein, "0.5-fold higher" refers to a 50% increase. For example a K_m of 3 would be 0.5-fold higher than a K_m of 2.

[0043] Advantageously, host cells engineered to express the mutant cellodextrin transporter showed improved cellodextrin consumption rates, higher ethanol yields, and higher ethanol productivity compared to host cells engineered to express a wild-type cellodextrin transporter lacking the mutation.

[0044] Accordingly, the present disclosure provides host cells containing, a recombinant polynucleotide encoding a mutant cellodextrin transporter, where the mutant cellodextrin transporter contains a transmembrane a-helix 1, a-helix 2, a-helix 3, a-helix 4, a-helix 5, a-helix 6, a-helix 7, a-helix 8, a-helix 9, a-helix 10, a-helix 11, a-helix 12, and a loop sequence positioned between a-helix 6 and a-helix 7, where the mutant cellodextrin transporter contains at least one mutation in the loop sequence, and where t the mutant cellodextrin transporter has a max that is at least 1-fold higher than the V_», of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence.

[0045] The present disclosure also provides methods of increasing transport of cellodextrin into a cell, by: providing a host cell of the present disclosure containing a recombinant polynucleotide encoding a mutant cellodextrin transporter; and culturing the cell in a medium such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared to a corresponding cell expressing a recombinant polynucleotide encoding a cellodextrin transporter lacking the at least one mutation in the loop sequence.

[0046] The present disclosure further provides isolated polypeptides having an amino acid sequence that is that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or at least 100% identical to SEQ ID NO: 2, wherein the polypeptide comprises an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 2; an amino acid sequence having the amino acid sequence SEQ ID NO: 2; isolated nucleic acids encoding such polypeptides; vectors containing such nucleic acids; and host cells containing such vectors. Polynucleotides Encoding Cellodextrin Transporters

[0047] Certain aspects of the present disclosure relate to host cells having increased rates of cellodextrin transport, where the increased rate of transport of the cellodextrin is the result of a recombinant polynucleotide encoding a mutant cellodextrin transporter containing at least one, at least two, or more mutations in the loop sequence. In some embodiments, the mutant cellodextrin transporter contains the amino acid sequence SEQ ID NO: 2. Such host cells may be used to increase the degradation of lignocellulosic biomass and to increase the rate of mixed sugar fermentation in the production of biofuels. In certain embodiments, the cellodextrin is selected from cellobiose, cellotriose, and cellotetraose. Other aspects of the present disclosure also relate to isolated polypeptides having the amino acid sequence of SEQ ID NO: 2, and to polynucleotides encoding such polypeptides.

[0048] As used herein, the terms "polynucleotide," "nucleic acid sequence," "sequence of nucleic acids," and variations thereof shall be generic to polydeoxyribonucleotides (containing 2- deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to any other type of

polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing non-nucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. Thus, these terms include known types of nucleic acid sequence modifications, for example, substitution of one or more of the naturally occurring nucleotides with an analog; inter- nucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters); those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); and those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.). As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC- IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970). [0049] As used herein, a "polypeptide" is an amino acid sequence containing a plurality of consecutive polymerized amino acid residues (e.g., at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues). In many instances, a polypeptide contains a polymerized amino acid residue sequence that is a transporter, a transcription factor, a predicted protein of unknown function, or a domain or portion or fragment thereof. A

transporter is involved in the movement of ions, small molecules, or macromolecules, such as a carbohydrate, across a biological membrane. A transcription factor can regulate gene expression and may increase or decrease gene expression in a host cell. The polypeptide optionally contains modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, and non-naturally occurring amino acid residues.

[0050] As used herein, "protein" refers to an amino acid sequence, oligopeptide, peptide, polypeptide, or portions thereof whether naturally occurring or synthetic.

Cellodextrin Transporters

[0051] Certain aspects of the present disclosure relate to polynucleotides encoding cellodextrin transporter polypeptides. In certain embodiments, the cellodextrin transporter is a mutant cellodextrin transporter. Suitable polynucleotides encoding cellodextrin transporter polypeptides that may be mutated include, without limitation, members of the Major Facilitator Superfamily sugar transporter family, such as HXT2.4, NCU00801, and NCU08114. Members of the Major Facilitator Superfamily (MFS) (Transporter Classification # 2.A.1) of transporters almost always contain 12 transmembrane a-helices, with an intracellular N- and C-terminus (S. S. Pao, I. T. Paulsen, M. H. Saier, Jr., Microbiol Mol Biol Rev 62, 1. Mar. 1998). While the primary sequence of MFS transporters varies widely, all are thought to share the tertiary structure of the E. coli lactose permease (LacY) (J. Abramson et al., Science 301, 610, Aug. 2003), and the E. coli Pi /glycerol-3-phospate (GlpT) (Y. Huang, M. J. Lemieux, J. Song, M. Auer, D. N. Wang, Science 301, 616 , Aug. 2003). In these examples the six N- and C-terminal helices form two distinct domains connected by a long cytoplasmic loop between helices 6 and 7. This symmetry corresponds to a duplication event thought to have given rise to the MFS. Substrate binds within a hydrophilic cavity formed by helices 1, 2, 4, and 5 of the N-terminal domain, and helices 7, 8, 10, and 11 of the C-terminal domain. This cavity is stabilized by helices 3, 6, 9, and 12.

[0052] The MFS sugar transporter family is defined by motifs found in transmembrane helices 6 and 12 [PESPR (SEQ ID NO: 11)/PETK (SEQ ID NO: 12)], and loops 2 and 8

(GRR/GRK) (M. C. Maiden, E. O. Davis, S. A. Baldwin, D. C. Moore, P. J. Henderson, Nature 325, 641, Feb. 1987). The entire Hidden Markov Model (HMM) for this family can be viewed at pfam.janelia.org/family/PF00083#tabview=tab3. PROSITE (N. Hulo et al, Nucleic Acids Res 34, D227, Jan. 2006) uses two motifs to identify members of this family. The first is

[LIVMSTAG] - [LIVMFSAG] - {SH} - {RDE} - [LIVMSA] - [DE] - {TD} - [LIVMFYWA] - G - R - [RK] - x(4,6) - [GSTA] (SEQ ID NO: 13). The second is [LIVMF] - x - G - [LIVMFA] - {V} - x - G - {KP} - x(7) - [LIFY] - x(2) - [EQ] - x(6) - [RK] (SEQ ID NO: 14). As an example of how to read a PROSITE motif, the following motif, [AC]-x-V-x(4)-{ED}, is translated as: [Ala or Cys]-any-Val-any-any-any-any-{ any but Glu or Asp} (SEQ ID NO: 15).

[0053] Moreover, cellodextrin transporters of the present disclosure may contain 12 transmembrane a-helices, a loop sequence between a-helix 6 and a-helix 7, and have N- and C- termini that are intracellular.

[0054] Additionally, in certain embodiments, transmembrane helix 1 contains the motif, [LIVM]-Y-[FL]-x(13)-[YF]-D (SEQ ID NO: 3); transmembrane helix 2 contains the motif, [YF]-x(2)-G-x(5)-[PVF]-x(6)-[DQ] (SEQ ID NO: 4); the loop connecting transmembrane helix 2 and transmembrane helix 3 contains the motif, G-R-[RK] (SEQ ID NO: 5); transmembrane helix 5 contains the motif, R-x(6)-[YF]-N (SEQ ID NO: 6); transmembrane helix 6 contains the motif, WR-[rVLA]-P-x(3)-Q (SEQ ID NO: 7); the sequence between transmembrane helix 6 and transmembrane helix 7 contains the motif, P-E-S-P-R-x-L-x(8)-A-x(3)-L-x(2)-Y-H (SEQ ID NO: 8); transmembrane helix 7 contains the motif, F-[GST]-Q-x-S-G-N-x-[LIV] (SEQ ID NO: 9); and transmembrane helix 10 and transmembrane helix 11 and the sequence between them contains the motif, L-x(3)-[YIV]-x(2)-E-x-L-x(4)-R-[GA]-K-G (SEQ ID NO: 10).

[0055] Accordingly, in certain embodiments, polynucleotides of the present disclosure encode a mutant cellodextrin transporter polypeptides containing 12 transmembrane a-helices {i.e., a-helix 1, a-helix 2, a-helix 3, a-helix 4, a-helix 5, a-helix 6, a-helix 7, a-helix 8, a-helix 9, α-helix 10, α-helix 11, α-helix 12), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight sequence motifs selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Additionally, the at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight sequence motifs may be combined in any number of combinations. For example, in certain embodiments, polynucleotides of the present disclosure encode a mutant cellodextrin transporter containing a-helix 1, a-helix 2, a-helix 3, a-helix 4, a- helix 5, a-helix 6, a-helix 7, a-helix 8, a-helix 9, a-helix 10, a-helix 11, a-helix 12, and the sequence motifs encoded by SEQ ID NO 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10.

[0056] Examples of suitable polynucleotides encoding cellodextrin transporter polypeptides that may be mutated to produce host cells with increased cellodextrin transport include, without limitation, cellodextrin transporters encoded by any of the genes listed in Table 1, homologs thereof, and orthologs thereof.

TABLE 1

Accession Number/Gene Name Organism

XP_964364.2/NCU00821 Neurospora crassa

XP_963898.1/NCU00988 Neurospora crassa

XP_961597.2/NCU01231 Neurospora crassa

XP_955927.2/NCU01494 Neurospora crassa

XP_959582.2/NCU02188 Neurospora crassa

XP_955977.1/NCU04537 Neurospora crassa

XP_959411.2/NCU04963 Neurospora crassa

XP_960481.1/NCU05519 Neurospora crassa

XP_959844.1/NCU05853 Neurospora crassa

XP_959888.1/NCU05897 Neurospora crassa

XP_960000.1/NCU06138 Neurospora crassa

XP_963873.1/NCU08114 Neurospora crassa

XP_958139.1/NCU09287 Neurospora crassa

XP_958069.2/NCU10021 Neurospora crassa

XP_962291.1/NCU07705 Neurospora crassa

XP_956635.1/NCU051379 Neurospora crassa

XP_956966.1/NCU01517 Neurospora crassa

XP_958905.1/NCU09133 Neurospora crassa

XP_957969.1/NCU10040 Neurospora crassa

XP_001220480 Chaetomium globusom CBS 148.51

XP_001912722 Podospora anserina

EEU41662 Nectria haematococca mpVI77-13-4

XP_660803 Aspergillus nidulans FGSC A4

XP_001218592 Aspergillus terreus NIH2624

XP_002341594 Talaromyces stipitatus ATCC 10500

XP_001395979 Aspergillus niger

XP_747891 Aspergillus fumigatus Af293

XP_00120996 Aspergillus terreus NIH2624

XP_001817400 Aspergillus oryzae RIB40

XP_001908539 Podospora anserina

XP_002568019 Penicillium chrysogenum Wisconsin 54-1255

XP_001209810 Aspergillus terreus NIH2624

XP_001820343 Aspergillus oryzae RIB40

XP_001210859 Aspergillus terreus NIH2624

XP_001728155 Neurospora crassa OR74A

XP_001826848 Aspergillus oryzae RIB40

XP_657617 Aspergillus nidulans FGSC A4

XP_002487579 Talaromyces stipitatus ATCC 10500

XP_001227497 Chaetomium globo sum CBS 148.51

215408 Trichoderma atroviridae

XP_001220290.1 Chaetomium globosum

ANID_08347 Aspergillus nidulans

51322 Pleurotus ostreatus Accession Number/Gene Name Organism

114107 Sporotrichum thermophile

XP_660418.1 Aspergillus nidulans

XP_364883.1 Magnaporthe grisea

XP_753099.1 Aspergillus fumigatus

211304 Trichoderma atroviridae

XP_001220469.1 Chaetomium globosum

63529 Tremella mesenterica

105952 Heterobasidion. annosum

252427 Cryphonectria parasitica

67752 Trichoderma ressei

XP_001268541.1 Aspergillus clavatus

77429 Neurospora discreta

3405 Trichoderma reesei

43941 Sporotrichum thermophile

XP .963801.1 Neurospora crassa

XP_001226269.1 Chaetomium globosum

46819 Trichoderma reesei

68287 Mycosphaerella graminicola

AFLA_000820A Aspergillus flavus

XP_002488227 Talaromyces stipitatus

XP_001400900 Aspergillus niger

XP_001220481 Chaetomium globosum CBS 148.51

XP_001912725 Podospora anserina

XP_660079 Aspergillus nidulans FGSC A4

AAL89823 Aspergillus niger

XP_002382573 Aspergillus flavus NRRL3357

XP_459386 Debaryomyces hansenii CBS767

XP_001825132 Aspergillus oryzae RIB40

XP_001389300 Aspergillus niger

XP_457508/DH61 Debaryomyces hansenii CBS767

XP_002551364 Candida tropicalis MYA-3404

XP_001523322 Loddewmyces elongisporus NRRL

XP_720384/29-4 Candida albicans SC5314

XP_456868 Debaryomyces hansenii CBS767

XP_001487429/29-6 Pichia guilliermondii ATCC 6260

XP_961039 Neurospora crassa

CAG88709/DH48 Debaryomyces hansenii CBS767

XP_001727326/29-9 Aspergillus oryzae

XP_001816757 Aspergillus oryzae

XP_002545773 Candida tropicalis MYA-3404

EEQ43601 Candida albicans WO-1

XP_001818631 Aspergillus oryzae RIB40

XP_002558275 Penicillium chrysogenum Wisconsin 54-1255

XP_001390883 Aspergillus niger Accession Number/Gene Name Organism

XP_750103 Aspergillus fumigatus Af293

XP_657854/32-8 Aspergillus nidulans FGSC A4

XP_001825068 Aspergillus oryzae RIB40

XP_681669/32- 10 Aspergillus nidulans FGSC A4

AB070824.1 Aspergillus oryzae

[0057] Accordingly, isolated polynucleotides and recombinant polynucleotides of the present disclosure may encode mutant cellodextrin transporter polypeptides derived from cellodextrin transporters having at least about 20%, or at least about 25%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100% amino acid sequence identity to the amino acid sequence of a cellodextrin transporter encoded by any of the genes listed in Table 1.

[0058] In certain embodiments, isolated polynucleotides of the present disclosure that are mutated to produce host cells with increased cellodextrin transport encode the cellodextrin transporter HXT2.4. The amino acid sequence of HXT2.4 is set forth in SEQ ID NO: 1.

HXT2.4 is a cellodextrin transporter derived from Schejfersomyces stipitis. HXT2.4 contains 12 transmembrane a-helices {i.e., a-helix 1, a-helix 2, a-helix 3, a-helix 4, a-helix 5, a-helix 6, a- helix 7, a-helix 8, a-helix 9, a-helix 10, a-helix 11, a-helix 12), and the sequence motifs encoded by SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10.

[0059] In some embodiments isolated polypeptides of the present disclosure encode a mutant cellodextrin transporter derived from a cellodextrin transporter having at least about 20%, or at least about 25%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or at least about 100% amino acid sequence identity to the amino acid sequence of HXT2.4. Homologous and orthologous cellodextrin transporters

[0060] Certain aspects of the present disclosure relate to isolated polynucleotides encoding mutant cellodextrin transporter polypeptides that are derived from homologs and/or orthologs of the cellodextrin transporters encoded by any of the genes listed in Table 1 above. "Homology" as used herein refers to sequence similarity between a reference sequence and at least a fragment of a second sequence. Homologs may be identified by any method known in the art, preferably, by using the BLAST tool to compare a reference sequence to a single second sequence or fragment of a sequence or to a database of sequences. As described below, BLAST will compare sequences based upon percent identity and similarity. "Orthology" as used herein refers to genes in different species that derive from a common ancestor gene.

[0061] The terms "identical" or percent "identity," in the context of two or more

polynucleotide or amino acid sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same {i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) in length.

[0062] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. When comparing two sequences for identity, it is not necessary that the sequences be contiguous, but any gap would carry with it a penalty that would reduce the overall percent identity. For blastn, the default parameters are Gap opening penalty=5 and Gap extension penalty=2. For blastp, the default parameters are Gap opening penalty=l 1 and Gap extension penalty=l .

[0063] A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions including, but not limited to from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol Biol 48(3):443-453, by the search for similarity method of Pearson and Lipman (1988) Proc Natl Acad Sci USA 85(8):2444-2448, by computerized

implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection [see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (Ringbou Ed)].

[0064] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the

neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89(22): 10915- 10919] alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0065] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, (1993) Proc Natl Acad Sci USA 90(12):5873- 5877). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a polynucleotide is considered similar to a reference sequence if the smallest sum probability in a comparison of the test polynucleotide to the reference polynucleotide is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0066] Other than percentage of sequence identity noted above, another indication that two polynucleotide sequences or polypeptides are substantially identical is that the polypeptide encoded by the first polynucleotide is immunologically cross-reactive with the antibodies raised against the polypeptide encoded by the second polynucleotide, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two

polynucleotide sequences are substantially identical is that the two molecules or their

complements hybridize to each other under stringent conditions, as described below. Yet another indication that two polynucleotide sequences are substantially identical is that the same primers can be used to amplify the sequence. [0067] Polynucleotides of the present disclosure may also include polynucleotides that encode conservatively modified variants of cellodextrin transporters encoded by the genes listed in Table 1 above. "Conservatively modified variants" as used herein include individual substitutions, deletions or additions to an encoded amino acid sequence which result 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 disclosure. The following eight groups 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)).

Mutated polynucleotides encoding cellodextrin transporters

[0068] In certain embodiments, recombinant polynucleotides encoding mutant cellodextrin transporters are produced by mutating a polynucleotide encoding a cellodextrin transporter of the present disclosure to increase the function and/or activity of the encoded cellodextrin transporter, compared to the function and/or activity of a corresponding cellodextrin transporter lacking such a mutation. In some embodiments, polynucleotides of the present disclosure contain at least one mutation that includes, without limitation, point mutations, missense mutations, substitution mutations, frameshift mutations, insertion mutations, duplication mutations, amplification mutations, translocation mutations, or inversion mutations that result in a polynucleotide encoding a cellodextrin transporter with increased function and/or activity.

[0069] Methods of generating at least one mutation in a polynucleotide of interest are well known in the art and include, without limitation, random mutagenesis and screening, site- directed mutagenesis, PCR mutagenesis, insertional mutagenesis, chemical mutagenesis, irradiation, and evolutionary engineering. It will be understood by one of skill in the art that certain described methods of generating at least one mutation in a polynucleotide of interest utilize a host organism or cell containing the polynucleotide of interest, while other described methods utilize a polynucleotide of interest that has been isolated from its host organism or cell, or that has been synthetically produced.

[0070] In other embodiments, polynucleotides of the present disclosure are modified to increase the function and/or activity of the encoded cellodextrin transporter. Polynucleotides of the present disclosure may be modified to contain one or more mutations that encode cellodextrin with increased function and/or activity. In one non-limiting example, HXT2.4 may be mutated to contain at least one amino acid substitution in the loop sequence encoded by amino acids 237-306 of SEQ ID NO: 1. Thus suitable polynucleotides homologous and/or orthologous to HXT2.4 may be mutated to encode an amino acid substitution at a loop sequence corresponding to amino acids amino acids 237-306 of SEQ ID NO: 1. The amino acid substitution may be any substitution that increases the function and/or activity of the encoded cellodextrin transporter.

[0071] Accordingly, in some embodiments, polynucleotides of the present disclosure encode a mutant cellodextrin transporter containing at least one mutation in a sequence corresponding to SEQ ID NO: 1. In other embodiments, polynucleotides of the present disclosure encode a mutant cellodextrin transporter containing at least one, at least two, or more mutations in a loop sequence corresponding to amino acids 237-306 of HXT2.4. Preferably, the at least one, at least two, or more mutations are amino acid substitutions. In certain aspects, the at least one, at least two, or more mutations are amino acid substitutions at a position corresponding to amino acid 291 of SEQ ID NO: 1. In further aspects, the at least one, at least two, or more mutations are selected from an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Arg amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Lys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Glu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Gin amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Cys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Phe amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Leu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Asn amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Thr amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Val amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, and combinations thereof. In other aspects, polynucleotides of the present disclosure encode mutant cellodextrin transporters that contain at least one, at least two, or more amino acid substitutions in regions other than the loop sequence corresponding to amino acids 237-306 of SEQ ID NO: 1 that increase the function and/or activity of the encoded cellodextrin transporter.

[0072] In some embodiments, the increased function and/or activity of a mutant cellodextrin transporter results in a host cell that transports cellodextrin at a rate faster than the rate of cellodextrin transport in a cell lacking the mutant cellodextrin transporter. For example, the rate of cellodextrin transport in a host cell containing a recombinant polynucleotide encoding a mutant cellodextrin transporter may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, or at least a higher percentage faster than the rate of cellodextrin transport in a host cell lacking the mutant cellodextrin transporter. Methods of measuring cellodextrin transport rate are known in the art and include the methods disclosed herein.

[0073] In certain embodiments, a mutant cellodextrin transporter of the present disclosure transports cellodextrin into a host cell of the present disclosure at maximal rate (i.e., VJ that is at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 0.6- fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1-fold, at least 1.25 fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5-fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the V„ of a corresponding non-mutant (i.e., wild type) cellodextrin transporter. Methods of measuring the V_», of a transporter, such as a cellodextrin transporter, are well known in the art and include those disclosed herein. For example, transport assays may be performed with GFP-tagged cellodextrin transporter polypeptides and tritium-labeled cellodextrin. [0074] In other embodiments, the concentration of a mutant cellodextrin transporter of the present disclosure at which the transporter transports cellodextrin into a host cell of the present disclosure at a rate that is half that of the V„ (i.e. , K_m) is at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8- fold, at least 0.9-fold, at least 1-fold, at least 1.25 fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5-fold, at least 4.75- fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the K_m of a corresponding non-mutant (i.e., wild type) cellodextrin transporter. Methods of measuring the K_m of a transporter, such as a cellodextrin transporter, are well known in the art and include those disclosed herein. For example, transport assays may be performed with GFP-tagged cellodextrin transporter polypeptides and tritium-labeled cellodextrin.

Preparation of polynucleotides

[0075] Sequences of the polynucleotides of the present disclosure are prepared by any suitable method known in the art, including, without limitation, direct chemical synthesis or cloning. For direct chemical synthesis, formation of a polymer of nucleic acids typically involves sequential addition of 3 '-blocked and 5 '-blocked nucleotide monomers to the terminal 5'-hydroxyl group of a growing nucleotide chain, wherein each addition is effected by nucleophilic attack of the terminal 5'-hydroxyl group of the growing chain on the 3 '-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. Such methodology is known to those of ordinary skill in the art and is described in the pertinent texts and literature [e.g., in Matteucci et al., (1980) Tetrahedron Lett 21:719-722; U.S. Pat. Nos. 4,500,707; 5,436,327; and 5,700,637]. In addition, the desired sequences may be isolated from natural sources by splitting DNA using appropriate restriction enzymes, separating the fragments using gel electrophoresis, and thereafter, recovering the desired polynucleotide sequence from the gel via techniques known to those of ordinary skill in the art, such as utilization of polymerase chain reactions (PCR; e.g., U.S. Pat. No. 4,683,195).

[0076] Each polynucleotide of the present disclosure can be incorporated into an expression vector. "Expression vector" or "vector" refers to a compound and/or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express polynucleotides and/or proteins other than those native to the cell, or in a manner not native to the cell. An "expression vector" contains a sequence of polynucleotides (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also includes materials to aid in achieving entry of the polynucleotide into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present disclosure include those into which a polynucleotide sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Preferred expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the polynucleotide sequence. Such plasmids, as well as other expression vectors, are well known in the art.

[0077] Incorporation of the individual polynucleotides may be accomplished through known methods that include, for example, the use of restriction enzymes (such as BamHI, EcoRI, Hhal, Xhol, Xmal, and so forth) to cleave specific sites in the expression vector, e.g., plasmid. The restriction enzyme produces single stranded ends that may be annealed to a polynucleotide having, or synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. Annealing is performed using an appropriate enzyme, e.g., DNA ligase. As will be appreciated by those of ordinary skill in the art, both the expression vector and the desired polynucleotide are often cleaved with the same restriction enzyme, thereby assuring that the ends of the expression vector and the ends of the polynucleotide are complementary to each other. In addition, DNA linkers maybe used to facilitate linking of polynucleotide sequences into an expression vector.

[0078] A series of individual polynucleotides can also be combined by utilizing methods that are known t in the art (e.g., U.S. Pat. No. 4,683,195).

[0079] For example, each of the desired polynucleotides can be initially generated in a separate PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences. When the PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and can act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are "spliced" together. In this way, a series of individual polynucleotides may be "spliced" together and subsequently transduced into a host cell simultaneously. Thus, expression of each of the plurality of polynucleotides is affected.

[0080] Individual polynucleotides, or "spliced" polynucleotides, are then incorporated into an expression vector. The present disclosure is not limited with respect to the process by which the polynucleotide is incorporated into the expression vector. Those of ordinary skill in the art are familiar with the necessary steps for incorporating a polynucleotide into an expression vector. A typical expression vector contains the desired polynucleotide preceded by one or more regulatory regions, along with a ribosome binding site, e.g., a nucleotide sequence that is 3-9 nucleotides in length and located 3-11 nucleotides upstream of the initiation codon in E. coli. See Shine and Dalgarno (1975) Nature 254(5495):34-38 and Steitz (1979) Biological Regulation and Development (ed. Goldberger, R. F.), 1:349-399 (Plenum, New York).

[0081] The term "operably linked" as used herein refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the DNA sequence or polynucleotide such that the control sequence directs the expression of a

polypeptide.

[0082] Regulatory regions include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired polynucleotide, thereby initiating transcription of the polynucleotide via an RNA polymerase enzyme. An operator is a sequence of polynucleotides adjacent to the promoter, which contains a protein-binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription initiates through the promoter. When present, the repressor protein specific to the protein-binding domain of the operator binds to the operator, thereby inhibiting transcription. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding repressor protein. Examples include, without limitation, lactose promoters (Lad repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator) and tryptophan promoters (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator). Another non-limiting example is the tac promoter (see de Boer et al., (1983) Proc Natl Acad Sci USA 80(l):21-25). As will be appreciated by those of ordinary skill in the art, these and other expression vectors may be used in the present disclosure, and the present disclosure is not limited in this respect.

[0083] Although any suitable expression vector may be used to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pSClOl, pBR322, pBBRlMCS-3, pUR, pEX, pMRlOO, pCR4, pBAD24, pUC19; and bacteriophages, such as Ml 3 phage and λ phage. Of course, such expression vectors may only be suitable for particular host cells. One of ordinary skill in the art, however, can readily determine through routine experimentation whether any particular expression vector is suited for any given host cell. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference may be made to the relevant texts and literature, which describe expression vectors and their suitability to any particular host cell.

Host Cells with Increased Cellodextrin Transport

[0084] Other aspects of the present disclosure relate to host cells containing a recombinant polynucleotide encoding a mutant cellodextrin transporter, where the mutant cellodextrin transporter has a V„ that is at least 1-fold higher than the V„ of a corresponding non-mutant (i.e., wild type) cellodextrin transporter. Further described herein are methods of increasing transport of cellodextrin into a host cell, methods of increasing growth of a host cell on a medium containing cellodextrin, methods of co-fermenting cellulose-derived and hemicellulose- derived sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a mutant cellodextrin transporter, where the host cell transports cellodextrin at a rate faster than the rate of cellodextrin transport in a cell lacking the mutant cellodextrin transporter. Further described herein are methods of increasing transport of a pentose into a host cell, methods of increasing growth of a host cell on a medium containing pentose sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a mutant cellodextrin transporter, where the host cell transports cellodextrin at a rate faster than the rate of cellodextrin transport in a cell lacking the mutant cellodextrin transporter.

[0085] "Host cell" and "host microorganism" are used interchangeably herein to refer to a living biological cell that can be transformed via insertion of recombinant DNA or RNA. Such recombinant DNA or RNA can be in an expression vector. Thus, a host organism or cell as described herein may be a prokaryotic organism (e.g., an organism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of ordinary skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.

[0086] Any prokaryotic or eukaryotic host cell may be used in the present disclosure so long as it remains viable after being transformed with a sequence of polynucleotides. Preferably, the host cell is not adversely affected by the transduction of the necessary polynucleotide sequences, the subsequent expression of the polypeptides (e.g., cellodextrin transporters), or the resulting intermediates. Suitable eukaryotic cells include, without limitation, fungal, plant, insect, and mammalian cells.

[0087] In preferred embodiments, the host is a fungal strain. "Fungi" as used herein includes, without limitation, the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).

[0088] In particular embodiments, the fungal host is a yeast strain. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of the present disclosure, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0089] In proffered embodiments, the yeast is oleaginous yeast, such as Rhodosporidium toruloides, Lipomyces starkyei, Yarrowia lipolytica, and Cryptococcus curvatus. [0090] In other preferred embodiments, the yeast host is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain.

[0091] In certain embodiments, the yeast host is a Saccharomyces carlsbergensis (Todkar, 2010), Saccharomyces cerevisiae (Duarte et al., 2009), Saccharomyces diastaticus,

Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces monacensis (GB-Analysts Reports, 2008), Saccharomyces bay anus (Kristen Publicover, 2010), Saccharomyces pastorianus (Nakao et al., 2007), Saccharomyces pombe (Mousdale, 2008), or Saccharomyces oviformis strain. In other preferred embodiments, the yeast host is

Kluyveromyces lactis (O.W. Merten, 2001), Kluyveromyces fragilis (Pestal et al., 2006; Siso, 1996), Kluyveromyces marxiamus (K. Kourkoutas et al., 2008), Pichia stipitis (Almeida et al., 2008), Candida shehatae (Ayhan Demirbas, 2003), or Candida tropicalis (Jamai et al., 2006). In other embodiments, the yeast host may be Yarrowia lipolytica (Biryukova E.N., 2009), Brettanomyces custersii (Spindler D.D. et al., 1992), or Zygosacchawmyces ux (Chaabane et al., 2006).

[0092] In another particular embodiment, the fungal host is a filamentous fungal strain. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally

characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0093] In preferred embodiments, the filamentous fungal host is, but not limited to, an

Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora,

Penicillium, Scytalidium, Thielavia, Tolypocladium, or Trichoderma strain.

[0094] In certain embodiments, the filamentous fungal host is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or

Aspergillus oryzae strain. In other embodiments, the filamentous fungal host is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum strain. In yet other preferred embodiments, the filamentous fungal host is a Humicola insolens, Humicola lanuginosa, Mucor miehei,

Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Scytalidium thermophilum, Sporotrichum thermophile (Topakas et al., 2003), or Thielavia terrestris strain. In a further embodiment, the filamentous fungal host is a Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain.

[0095] In other preferred embodiments, the host cell is prokaryotic, and in certain embodiments, the prokaryotes are E. coli (Dien, B.S. et al., 2003; Yomano, L. P. et al., 1998; Moniruzzaman et al., 1996), Bacillus subtilis (Susana Romero et al., 2007), Zymomonas mobilis (B. S. Dien et al, 2003; Weuster Botz, 1993; Alterthum and Ingram, 1989), Clostridium sp. (Zeikus, 1980; Lynd et al., 2002; Demain et al., 2005), Clostridium phytofermentans (Leschine S., 2010), Clostridium thermocellum (Lynd et al., 2002), Clostridium beijerinckii (Giles Clark, 2008), Clostridium acetobutylicum (Moorella thermoacetica) (Huang W.C. et al., 2004;

Dominik et al., 2007), Thermoanaerobacterium sac char olyticum (Marietta Smith, 2009), or Klebsiella oxytoca (Dien, B.S. et al., 2003; Zhou et al., 2001; Brooks and Ingram, 1995). In other embodiments, the prokaryotic host cells are Carboxydocella sp. (Dominik et al., 2007), Corynebacterium glutamicum (Masayuki Inui, et al., 2004), Enterobacteriaceae (Ingram et al., 1995), Erwinia chrysanthemi (Zhou and Ingram, 2000; Zhou et al., 2001), Lactobacillus sp. (McCaskey, T.A., et al., 1994), Pediococcus acidilactici (Zhou, S. et al., 2003),

Rhodopseudomonas capsulata (X.Y. Shi et al., 2004), Streptococcus lactis (J.C. Tang et al., 1988), Vibrio furnissii (L.P. Wackett, 2010), Vibrio furnissii Ml (Park et al, 2001),

Caldicellulosiruptor saccharolyticus (Z. Kadar et al., 2004), or Xanthomonas campestris (S.T. Yang et al., 1987). In other embodiments, the host cells are cyanobacteria. Additional examples of bacterial host cells include, without limitation, those species assigned to the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, Synechococcus, Synechocystis, and Paracoccus taxonomical classes. [0096] In especially preferred embodiments, the host cell is selected from Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus,

Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe,

Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Myceliophthora thermophila, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium

acetobutylicum, Moorella thermoacetica, Escherichia coli, Klebsiella oxytoca,

Thermoanaerobacterium sac char olyticum, Bacillus subtilis, Rhodosporidium toruloides, Lipomyces starkyei, Yarrowia lipolytica, and Cryptococcus curvatus. Saccharomyces sp. may include Industrial Saccharomyces strains. Argueso et al. discuss the genome structure of an Industrial Saccharomyces strain commonly used in bioethanol production as well as specific gene polymorphisms that are important for bioethanol production (Genome Research, 19: 2258- 2270, 2009).

[0097] The host cells of the present disclosure may be genetically modified in that recombinant polynucleotides have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing one or more polynucleotide constructs encoding one or more polypeptides for different functions.

[0098] "Recombinant polynucleotide" or "heterologous polynucleotide" as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to (i.e., not naturally found in) a given host cell; (b) the sequence may be naturally found in a given host cell, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids contains two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Specifically, the present disclosure describes the introduction of an expression vector into a host cell, wherein the expression vector contains a polynucleotide sequence coding for a polypeptide that is not normally found in a host cell or contains a polynucleotide coding for a polypeptide that is normally found in a cell but is under the control of different regulatory sequences. With reference to the host cell's genome, then, the nucleic acid sequence that codes for the polypeptide is recombinant.

[0099] In some embodiments, the host cell naturally produces any of the polypeptides encoded by the polynucleotides of the present disclosure. The genes encoding the desired polypeptides may be heterologous to the host cell or these genes may be endogenous to the host cell but are operatively linked to heterologous promoters and/or control regions which result in the higher expression of the gene(s) in the host cell. In other embodiments, the host cell does not naturally produce the desired polypeptides, and contains heterologous polynucleotide constructs capable of expressing one or more genes necessary for producing those molecules.

[0100] "Endogenous" as used herein with reference to a polynucleotide molecule or polypeptide and a particular cell or microorganism refers to a polynucleotide sequence or peptide that is in the cell and was not introduced into the cell using recombinant engineering techniques; for example, a gene that was present in the cell when the cell was originally isolated from nature.

[0101] "Genetically engineered" or "genetically modified" refer to any recombinant DNA or RNA method used to create a prokaryotic or eukaryotic host cell that expresses a polypeptide at elevated levels, at lowered levels, or in a mutated form. In other words, the host cell has been transfected, transformed, or transduced with a recombinant polynucleotide molecule, and thereby been altered so as to cause the cell to alter expression of a desired polypeptide. Methods and vectors for genetically engineering host cells are well known in the art; for example various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly updates). Genetically engineering techniques include but are not limited to expression vectors, targeted homologous recombination and gene activation (see, for example, U.S. Pat. No. 5,272,071 to Chappel) and trans-activation by engineered transcription factors (see, for example, Segal et al., (1999) Proc Natl Acad Sci USA 96(6):2758-2763).

[0102] Genetic modifications that result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene. More specifically, reference to increasing the action (or activity) of enzymes or other proteins discussed herein generally refers to any genetic modification of the host cell in question which results in increased expression and/or functionality (biological activity) of the enzymes or proteins and includes higher activity or action of the proteins (e.g. , specific activity or in vivo enzymatic activity), reduced inhibition or degradation of the proteins, and overexpression of the proteins. For example, gene copy number can be increased, expression levels can be increased by use of a promoter that gives higher levels of expression than that of the native promoter, or a gene can be altered by genetic engineering or classical mutagenesis to increase the biological activity or function of a protein. Combinations of some of these modifications are also possible.

[0103] Genetic modifications which result in a decrease in gene expression, in the function of the gene, or in the function of the gene product (i.e. , the polypeptide encoded by the gene) can be referred to as inactivation (complete or partial), deletion, interruption, blockage, silencing, or down-regulation, or attenuation of expression of a gene. For example, a genetic modification in a gene which results in a decrease in the function of the polypeptide encoded by such gene, can be the result of a complete deletion of the gene (i.e. , the gene does not exist, and therefore the polypeptide does not exist), a mutation in the gene which results in incomplete or no translation of the polypeptide (e.g. , the polypeptide is not expressed), or a mutation in the gene which decreases or abolishes the natural function of the polypeptide (e.g. , a polypeptide is expressed which has decreased or no enzymatic activity or action). More specifically, reference to decreasing the action of a polypeptide generally refers to any genetic modification in the host cell in question, which results in decreased expression and/or functionality (biological activity) of the polypeptides and includes decreased activity of polypeptides (e.g. , decreased transport), increased inhibition or degradation of the polypeptides as well as a reduction or elimination of expression of the polypeptides. Combinations of some of these modifications are also possible. Blocking or reducing the production of a polypeptide can include placing the gene encoding the polypeptide under the control of a promoter that requires the presence of an inducing compound in the growth medium. By establishing conditions such that the inducer becomes depleted from the medium, the expression of the gene encoding the polypeptide (and therefore, of polypeptide synthesis) could be turned off. Blocking or reducing the action of a polypeptide could also include using an excision technology approach similar to that described in U.S. Pat. No.

4,743,546, incorporated herein by reference. To use this approach, the gene encoding a polypeptide of interest is cloned between specific genetic sequences that allow specific, controlled excision of the gene from the genome. Excision could be prompted by, for example, a shift in the cultivation temperature of the culture, as in U.S. Pat. No. 4,743,546, or by some other physical or nutritional signal.

[0104] In general, according to the present disclosure, an increase or a decrease in a given characteristic of a mutant or modified polypeptide is made with reference to the same

characteristic of a wild- type (i.e. , normal, not modified) polypeptide that is derived from the same organism (from the same source or parent sequence), which is measured or established under the same or equivalent conditions. Similarly, an increase or decrease in a characteristic of a genetically modified host cell (e.g. , expression and/or biological activity of a polypeptide, or production of a product) is made with reference to the same characteristic of a wild-type host cell of the same species, and preferably the same strain, under the same or equivalent conditions. Such conditions include the assay or culture conditions (e.g. , medium components, temperature, pH, etc.) under which the activity of the polypeptide (e.g. , expression or biological activity) or other characteristic of the host cell is measured, as well as the type of assay used, the host cell that is evaluated, etc. As discussed above, equivalent conditions are conditions (e.g. , culture conditions) which are similar, but not necessarily identical (e.g. , some conservative changes in conditions can be tolerated), and which do not substantially change the effect on cell growth or enzyme expression or biological activity as compared to a comparison made under the same conditions.

[0105] Preferably, a genetically modified host cell that has a genetic modification that increases or decreases the activity or function of a given polypeptide (e.g., a transporter, an enzyme) has an increase or decrease, respectively, in the activity or action (e.g. , expression, production and/or biological activity) of the polypeptide, as compared to the activity of the wild- type polypeptide in a wild-type host cell, of at least about 5%, and more preferably at least about 10%, and more preferably at least about 15%, and more preferably at least about 20%, and more preferably at least about 25%, and more preferably at least about 30%, and more preferably at least about 35%, and more preferably at least about 40%, and more preferably at least about 45%, and more preferably at least about 50%, and more preferably at least about 55%, and more preferably at least about 60%, and more preferably at least about 65%, and more preferably at least about 70%, and more preferably at least about 75%, and more preferably at least about 80%, and more preferably at least about 85%, and more preferably at least about 90%, and more preferably at least about 95%, or any percentage, in whole integers between 5% and 100% (e.g., 6%, 7%, 8%, etc.). The same differences are preferred when comparing an isolated modified polynucleotide molecule or polypeptide directly to the isolated wild-type polynucleotide molecule or polypeptide (e.g., if the comparison is done in vitro as compared to in vivo).

[0106] In another aspect of the present disclosure, a genetically modified host cell that has a genetic modification that increases or decreases the activity of a given polypeptide (e.g., a transporter, an enzyme) has an increase or decrease, respectively, in the activity or action (e.g., expression, production and/or biological activity) of the polypeptide, as compared to the activity of the wild-type polypeptide in a wild-type host cell, of at least about 2-fold, and more preferably at least about 5-fold, and more preferably at least about 10-fold, and more preferably about 20- fold, and more preferably at least about 30-fold, and more preferably at least about 40-fold, and more preferably at least about 50-fold, and more preferably at least about 75-fold, and more preferably at least about 100-fold, and more preferably at least about 125-fold, and more preferably at least about 150-fold, or any whole integer increment starting from at least about 2- fold (e.g., 3-fold, 4-fold, 5-fold, 6-fold, etc.).

Mutant Cellodextrin Transporters

[0107] In one aspect, host cells of the present disclosure contain a recombinant

polynucleotide encoding a mutant cellodextrin transporter containing transmembrane a-helix 1, a-helix 2, a-helix 3, a-helix 4, a-helix 5, a-helix 6, a-helix 7, a-helix 8, a-helix 9, a-helix 10, a- helix 11, a-helix 12, and a loop sequence positioned between a-helix 6 and a-helix 7, where the mutant cellodextrin transporter contains at least one, at least two, or more mutations in the loop sequence, and where the mutant cellodextrin transporter has a V„ that is at least 1-fold higher than the V_», of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence. In some embodiments, the loop sequence corresponds to amino acids 237-306 of HXT2.4. In other embodiments, the at least one, at least two, or more mutations are an amino acid substitution. In still other embodiments, the at least one, at least two, or more mutations are an amino acid substitutions at a position corresponding to amino acid 291 of HXT2.4.

Preferably, the at least one, at least two, or more mutations are selected from an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Arg amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Lys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Glu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Gin amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Cys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Phe amino acid substitution at a position

corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Leu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Asn amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Thr amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Val amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, and combinations thereof.

[0108] In some embodiments, host cells of the present disclosure may further contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight sequence motifs selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Additionally, the at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight sequence motifs may be combined in any number of combinations. For example, a host cell of the present disclosure may contain a recombinant polynucleotide encoding a mutant cellodextrin transporter containing a-helix 1, a-helix 2, a-helix 3, a-helix 4, a-helix 5, a-helix 6, a-helix 7, a- helix 8, a-helix 9, a-helix 10, a-helix 11, a-helix 12, and five sequence motifs encoded by SEQ ID NO 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10. Alternatively, a host cell of the present disclosure may contain a recombinant polynucleotide encoding a mutant cellodextrin transporter containing a-helix 1, a-helix 2, a-helix 3, a-helix 4, a-helix 5, a-helix 6, a-helix 7, a-helix 8, a-helix 9, a-helix 10, a-helix 11, a-helix 12, and all eight sequence motifs encoded by SEQ ID NOs: 3-10.

[0109] In other embodiments, a host cell of the present disclosure contains a recombinant polynucleotide encoding a mutant cellodextrin transporter having an amino acid sequence that is at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of wild- type HXT2.4 (i.e., SEQ ID NO: 1) or a mutant HXT2.4 having an Ala to Asp substitution at position 291 (i.e., SEQ ID NO: 2).

[0110] In some embodiments, a host cell of the present disclosure contains a recombinant polynucleotide encoding a mutant cellodextrin transporter having a V_», that is at least 0.1 -fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7- fold, at least 0.8-fold, at least 0.9-fold, at least 1-fold, at least 1.25 fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5- fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the V„ of a corresponding non-mutant (i.e., wild type) cellodextrin transporter.

[0111] In other embodiments, a host cell of the present disclosure contains a recombinant polynucleotide encoding a mutant cellodextrin transporter having a K_m that is at least 0.1 -fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7- fold, at least 0.8-fold, at least 0.9-fold, at least 1-fold, at least 1.25 fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5- fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the K_m of a corresponding non-mutant (i.e., wild type) cellodextrin transporter.

[0112] In some embodiments, a host cell of the present disclosure containing a recombinant polynucleotide encoding a mutant cellodextrin transporter exhibits a rate of growth that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5- fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5-fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the growth rate exhibited by a corresponding cell lacking the mutant cellodextrin transporter. Methods of measuring cell growth rate are well known in the art and include those disclosed herein.

[0113] In other embodiments, a host cell of the present disclosure containing a recombinant polynucleotide encoding a mutant cellodextrin transporter exhibits a cellodextrin consumption rate that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5-fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the cellodextrin consumption rate exhibited by a corresponding cell lacking the mutant cellodextrin transporter. Methods of measuring the rate of cellodextrin consumption are well known in the art and include those disclosed herein.

[0114] In further embodiments, a host cell of the present disclosure containing a recombinant polynucleotide encoding a mutant cellodextrin transporter produces a biofuel, such as ethanol, with a biofuel (e.g. , ethanol) productivity that is at least 0.2-fold, at least 0.25-fold, at least 0.3- fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85- fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5- fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the biofuel (e.g. , ethanol) productivity exhibited by a corresponding cell lacking the mutant cellodextrin transporter. As used herein, "biofuel productivity" refers to the amount of biofuel, such as ethanol, produced per unit time by host a cell of the present disclosure. Methods of measuring biofuel productivity are well known in the art and include those disclosed herein.

Cellodextrin Phosphorylases

[0115] In further aspects, host cells of the present disclosure may also contain one or more polynucleotides encoding at least a catalytic domain of a cellodextrin phosphorylase.

Cellodextrin phosphorylases of the present disclosure catalyze the degradation of a cellodextrin by utilizing inorganic phosphate to cleave β-glucosidic linkages between glucose moieties in the cellodextrin. Cellodextrin phosphorylases of the present disclosure may include polypeptides having EC 2.4.1.49 activity, which catalyze the following reaction: (l,4-P-D-glucosyl)_n + inorganic phosphate

+ a-D-glucose-1 -phosphate. Polypeptides having EC 2.4.1.49 activity belong to the GH 94 family of glycoside hydrolases. Polypeptides with EC 2.4.1.49 activity include, without limitation, l,4-beta-D-oligo-D-glucan:phosphate alpha-D- glucosyltransferases and beta-l,4-oligoglucan:orthophosphate glucosyltransferases.

[0116] Cellodextrin phosphorylases of the present disclosure may also include polypeptides having EC 2.4.1.20 activity, which catalyze the following reaction: cellobiose + inorganic phosphate a-D-glucose-1 -phosphate + D-glucose. Polypeptides having EC 2.4.1.20 activity belong to the hexosyltransferase family of glycoside hydrolases. Polypeptides with EC 2.4.1.20 activity include, without limitation, cellobiose phosphorylases and cellobiose:phosphate alpha- D-glucosyltransferases.

[0117] As used herein, a catalytic domain of a cellodextrin phosphorylase is any domain that catalyzes the cleavage of beta-glucosidic linkage between glucose moieties in cellodextrins with release of glucose- 1 phosphate. The polynucleotide encoding a catalytic domain of cellodextrin phosphorylase may be endogenous or heterologous to the host cell. In certain embodiments, the catalytic domain of the cellodextrin phosphorylase is located intracellularly in the host cell.

[0118] Cellodextrin phosphorylases can be identified by the PROSITE motif: G-x(2)-[FY]-x- N- [AGS] -x- [AS] - W- [APS] -V- [IL] - [ AS] -x(2)- A-x(2)- [DE] -x- [ AI] -x(3)- [LM V] - [DEN] - [ AS V] - [ILV]-x(3)-L-x-T-x(2)-G-[ILV]-x(2)-[SV]-x-P-[AG] (SEQ ID NO: 26). Cellodextrin

phosphorylases having cellobiose phosphorylase activity {i.e., cellobiose phosphorylases) can be identified by the PROSITE motif: Y-Q-[CN]-M-[IV]-T-F-[CN]-[FILMV]-[AS]-R-[ST]-[AS]-S- [FY] -[FY] -E-[STV] -G-x- [GS] -R-G- [IM] -G-F-R-D-S- [ACNS] -Q-D- [ILV] - [ILMV] -G-x- V-H-x- [IV]-P-[ADEST]-x-[AV]-[KR]-[AEQ]-x-[IL]-[FIL]-D (SEQ ID NO: 27). As an example of how to read a PROSITE motif, the following motif, [AC]-x-V-x(4)-{ED}, is translated as: [Ala or Cys]-any-Val-any-any-any-any-{any but Glu or Asp}. Examples of suitable cellodextrin phosphorylases, including cellobiose phosphorylases, include, without limitation, those disclosed in International Patent Application No. PCT/US2012/024186. Preferably, the cellodextrin phosphorylase is a Clostridium lentocellum cellodextrin phosphorylase (CDP), a Clostridium thermocellum CDP, an Acidovibrio cellulolyticus CDP, a Celvibrio gilvus cellobiose

phosphorylase (CBP), a Saccharophagus degradans CBP, or a Clostridium thermocellum CBP.

[0119] In some embodiments, the cellodextrin phosphorylase has an amino acid sequence that has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to the amino acid sequence of SEQ ID NO: 20 (CDP_Clent), SEQ ID NO: 21 (CDP_Ctherm), or SEQ ID NO: 22

(CDP_Acell). In other embodiments, the cellodextrin phosphorylase has cellobiose

phosphorylase activity. In certain embodiments, the cellodextrin phosphorylase with cellobiose phosphorylase activity has an amino acid sequence that has at least 29%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to the amino acid sequence of SEQ ID NO: 23 (CgCBP), SEQ ID NO: 24 (SdCBP), or SEQ ID NO: 25 (CtCBP). β-Glucosidases

[0120] In other aspects, host cells of the present disclosure further contain a recombinant polynucleotide that encodes at least a catalytic domain of a β-glucosidase. As used herein, "β- glucosidase" refers to a β-D-glucoside glucohydrolase (E.C. 3.2.1.21) enzyme that catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues with the release of β-D-glucose. A catalytic domain of β-glucosidase has β-glucosidase activity as determined, for example, according to the basic procedure described by Venturi et al., 2002. A catalytic domain of a β- glucosidase is any domain that catalyzes the hydrolysis of terminal non-reducing residues in β- D-glucosides with release of glucose. In preferred embodiments, the β-glucosidase is a glycosyl hydrolase family 1 member. Members of this group can be identified by the motif,

[LIVMFSTC] - [LIVFYS] - [LIV] - [LIVMST] - E - N - G - [LIVMFAR] - [CSAGN] (SEQ ID NO: 16). Here, E is the catalytic glutamate (webpage expasy.org/cgi-bin/prosite-search- ac?PDOC00495). In certain embodiments, the polynucleotide encoding a catalytic domain of β- glucosidase is heterologous to the host cell. In certain embodiments, the catalytic domain of β- glucosidase is located intracellularly in the host cell. In some embodiments, the β-glucosidase is from N. crassa. Preferably, the β-glucosidase is NCU00130. In certain embodiments, the β- glucosidase is an ortholog of NCU00130. Examples of orthologs of NCU00130 include, without limitation, T. melanosporum, CAZ82985.1; A. oryz e, BAE57671.1; P. placenta, EED81359.1; P. chrysosporium, BAE87009.1; Kluyveromyces lactis, CAG99696.1; Laccaria bicolor, EDR09330; Clavispora lusitaniae, EEQ37997.1; and Pichia stipitis, ABN67130.1. Other β-glucosidases that may be used include those from the glycosyl hydrolase family 3. These β- glucosidases can be identified by the following motif according to PROSITE: [LIVM](2) - [KR] - x - [EQKRD] - x(4) - G - [LIVMFTC] - [LIVT] - [LIVMF] - [ST] - D - x(2) - [SGADNIT] (SEQ ID NO: 17). Here D is the catalytic aspartate. Typically, any β-glucosidase may be used that contains the conserved domain of β-glucosidase/6-phospho-β-glucosidase/β-galactosidase found in NCBI sequence COG2723. Catalytic domains from specific β-glucosidases may be preferred depending on the cellodextrin transporter contained in the host cell.

Pentose Utilization

[0121] In other aspects, host cells of the present disclosure further contain one or more polynucleotides encoding one or more enzymes involved in pentose utilization. The one or more polynucleotides may be endogenous or heterologous to the host cell. "Pentose", as used herein, refers to any monosaccharide with five carbon atoms. Examples of pentoses include, without limitation, xylose, arabinose, mannose, galactose, and rhamnose. The one or more enzymes involved in pentose utilization may include, for example, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabitinol 4- dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase in any combination. These enzymes may come from any organism that naturally metabolizes pentose sugars. Examples of such organisms include, for example, Kluyveromyces sp., Zymomonas sp., E. coli, Clostridium sp., and Pichia sp.

[0122] In embodiments where the host cell is a Saccharomyces sp., preferred pentose utilizing strains include DA24-16 and L2612. Other host cells containing polynucleotides encoding enzymes involved in pentose utilization include a DuPont Zymomonas strain (WO 2009/058927) and a Saccharomyces strain (US 5,789,210).

[0123] In further embodiments, host cells of the present disclosure also contain a

recombinant polynucleotide encoding a pentose transporter. Suitable pentose transporters include, without limitation, NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, and XUT3 {e.g., see U.S. Patent Application Publication No. US 2011/0020910).

Additionally, suitable pentose transporters may also include, without limitation, Gxsl from C. intermedia, Autl from P. stipitis, Xylhp from D. hansenii (Nobre et al., 1999), xylose transporter from K. marxianus (Stambuk et al., 2003), LAT1 and LAT2 from Ambrosiozyma monospora (EMBL AY923868 and AY923869, respectively, R. Verho et al.), ART1 from C.

arabinofermentans (Fonseca et al., 2007), KmLATl from K. marxiamus (Knoshaug et al., 2007), PgLAT2 from P. guilliermondii (Knoshaug et al., 2007), and araT from P. stipitis (Boles & Keller, 2008).

Methods of Producing and Culturing Host Cells with Increased Cellodextrin Transport

[0124] Further aspects of the present disclosure relate to the production of host cells containing recombinant polynucleotides encoding mutant cellodextrin transporters that allow the cell to transport cellodextrin at a rate faster than the rate of cellodextrin transport in a cell lacking the mutant cellodextrin transporters. Further described herein are methods of increasing transport of cellodextrin into a host cell, methods of increasing growth of a host cell on a medium containing cellodextrin, methods of co-fermenting cellulose-derived and hemicellulose- derived sugars, and methods of making hydrocarbons or hydrocarbon derivatives by providing a host cell containing a recombinant polynucleotide encoding a mutant cellodextrin transporter.

[0125] Methods of producing and culturing host cells of the present disclosure may include the introduction or transfer of expression vectors containing the recombinant polynucleotides of the present disclosure into a host cell. Such methods for transferring expression vectors into host cells are well known in the art. For example, one method for transforming E. coli with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation {i.e., the application of current to increase the permeability of cells to polynucleotide sequences) may be used to transfect the host cell. Also, microinjection of the polynucleotide sequences provides the ability to transfect host cells. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.

[0126] The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host, or a transposon may be used.

[0127] The vectors preferably contain one or more selectable markers which permit easy selection of transformed hosts. A selectable marker is a gene the product of which provides, for example, biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selection of bacterial cells may be based upon antimicrobial resistance that has been conferred by genes such as the amp, gpt, neo, and hyg genes.

[0128] Suitable markers for yeast hosts include, without limitation, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host include, without limitation, amdS (acetamidase), argB (ornithine carbamoyltransf erase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyl transferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in Aspergillus are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of

Streptomyces hygroscopicus. Preferred for use in Trichoderma are bar and amdS.

[0129] The vectors preferably contain an element(s) that permits integration of the vector into the host's genome or autonomous replication of the vector in the cell independent of the genome.

[0130] For integration into the host genome, the vector may rely on the gene's sequence or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host. The additional nucleotide sequences enable the vector to be integrated into the host genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host.

Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host by non-homologous recombination.

[0131] For autonomous replication, the vector may further include an origin of replication enabling the vector to replicate autonomously in the host in question. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a sequence that enables a plasmid or vector to replicate in vivo. Examples of origins of replication for use in a yeast host are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991; Cullen et al., 1987; WO 00/24883).

Isolation of the AMA1 gene and construction of plasmids or vectors containing the gene can be accomplished according to the methods disclosed in WO 00/24883. [0132] For other hosts, transformation procedures may be found, for example, in Jeremiah D. Read, et al., Applied and Environmental Microbiology, Aug. 2007, p. 5088-5096, for

Kluyveromyces, in Osvaldo Delgado, et al., FEMS Microbiology Letters 132, 1995, 23-26, for Zymomonas, in US 7,501,275 for Pichia stipitis, and in WO 2008/040387 for Clostridium.

[0133] More than one copy of a gene may be inserted into the host to increase production of the gene product. An increase in the copy number of the gene can be obtained by integrating at least one additional copy of the gene into the host genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the gene, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0134] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present disclosure are well known in the art (see, e.g., Sambrook et al., 1989, supra).

[0135] The host cell is transformed with at least one expression vector. When only a single expression vector is used (without the addition of an intermediate), the vector will contain all of the polynucleotide sequences necessary.

[0136] Once the host cell has been transformed with the expression vector, the host cell is allowed to grow. Methods of the present disclosure may include culturing the host cell such that recombinant polynucleotides in the cell are expressed. For microbial hosts, this process entails culturing the cells in a suitable medium. Typically cells are grown at 35°C in appropriate media. Preferred growth media in the present disclosure include, for example, common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular host cell will be known by someone skilled in the art of microbiology or fermentation science. Temperature ranges and other conditions suitable for growth are known in the art (e.g., Bailey and Ollis 1986).

[0137] According to some aspects of the present disclosure, the culture media contains a carbon source for the host cell. Such a "carbon source" generally refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. These include, for example, various monosaccharides such as glucose, oligosaccharides, polysaccharides, a biomass polymer such as cellulose or hemicellulose, xylose, arabinose, disaccharides, such as sucrose, saturated or unsaturated fatty acids, succinate, lactate, acetate, ethanol, etc., or mixtures thereof. The carbon source can additionally be a product of photosynthesis, including, but not limited to glucose.

[0138] In preferred embodiments, the carbon source is a biomass polymer such as cellulose or hemicellulose. As described herein, a "biomass polymer" is any polymer contained in biological material. The biological material may be living or dead. A biomass polymer includes, for example, cellulose, xylan, xylose, hemicellulose, lignin, mannan, and other materials commonly found in biomass. Non-limiting examples of sources of a biomass polymer include, without limitation, plant material, municipal solid waste, and wastepaper. Plant material may include, without limitation, Miscanthus, energy grass, elephant grass, switchgrass, cord grass, rye grass, reed canary grass, common reed, wheat straw, barley straw, canola straw, oat straw, corn stover, soybean stover, oat hulls, oat spelt, sorghum, rice hulls, sugarcane bagasse, corn fiber, barley, oats, flax, wheat, linseed, citrus pulp, cottonseed, groundnut, rapeseed, jute, hemp, bamboo, sisal, abaca, sunflower, peas, lupines, palm kernel, coconut, konjac, locust bean gum, gum guar, soy beans, Distillers Dried Grains with Solubles (DDGS), Blue Stem, corncobs, pine, conifer softwood, eucalyptus, birchwood, willow, aspen, poplar wood, hybrid poplar, energy cane, short-rotation woody crop, crop residue, yard waste, distillers grains, legume plants, sugar beet pulp, wood chips, sawdust, biomass crops (e.g., Crambe), and combinations thereof.

[0139] In addition to an appropriate carbon source, media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathways necessary for the fermentation of various sugars and the production of hydrocarbons and hydrocarbon derivatives. Reactions may be performed under aerobic or anaerobic conditions where aerobic, anoxic, or anaerobic conditions are preferred based on the requirements of the microorganism. As the host cell grows and/or multiplies, expression of the enzymes, transporters, or other polypeptides necessary for growth on various sugars or biomass polymers, sugar fermentation, or synthesis of hydrocarbons or hydrocarbon derivatives is affected.

Methods of Increasing Transport of a Sugar into a Cell

[0140] The present disclosure provides methods of increasing transport of a sugar, such as a cellodextrin, into a host cell. In one aspect, the present disclosure provides a method of transporting cellodextrin into a cell, including a first step of providing a host cell of the present disclosure containing a recombinant polynucleotide encoding a mutant cellodextrin transporter, and a second step of culturing the cell such that the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared to a corresponding cell expressing a recombinant polynucleotide encoding a cellodextrin transporter lacking the at least one, at least two, or more mutations in the loop sequence. Any host cell of the present disclosure as described in the section entitled "Host Cells with Increased Cellodextrin Transport" may be used. Transport of cellodextrin into a cell may be measured by any method known to one of skill in the art, including measuring uptake of [ H]- cellobiose into cells or measuring the ability of an S. cerevisiae host cell to grow when cellobiose is the sole carbon source. Typically, the host cell containing the recombinant polynucleotide and the host cell that does not contain the recombinant polynucleotide will otherwise be identical in genetic background.

[0141] In methods of increasing transport of cellodextrin into a cell, the cell may be cultured in a medium containing a cellulase-containing enzyme mixture from an altered organism, where the mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. The organism may be altered to reduce the expression of β-glucosidase, such as by mutation of a gene encoding β-glucosidase or by targeted RNA interference or the like.

[0142] In certain embodiments, the host cell also contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. The one or more enzymes may be, for example, L-arabinose isomerase, L- ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L- arabitinol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, or any other pentose utilization enzymes known in the art.

[0143] In other embodiments, the cellodextrin is transported into the cell at a maximal rate (i.e., Ymax) that is at least at least 0.1-fold, at least 0.2-fold, at least 0.3-fold, at least 0.4-fold, at least 0.5-fold, at least 0.6-fold, at least 0.7-fold, at least 0.8-fold, at least 0.9-fold, at least 1-fold, at least 1.25 fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5- fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5-fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the V_», of cellodextrin transport into a corresponding cell having a corresponding cellodextrin transporter lacking the at least one, at least two, or more mutations in the loop sequence.

[0144] In still other embodiments, the cellodextrin is consumed at a rate that is at least 0.2- fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75- fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4- fold, at least 4.25 fold, at least 4.5-fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the rate of cellodextrin consumption by a corresponding cell lacking the mutant cellodextrin transporter.

[0145] The methods of increasing transport of cellodextrin into a host cell may further include a step of producing a fermentation product by culturing the host cell under conditions sufficient to ferment the cellodextrin. Culturing conditions sufficient to ferment cellodextrin are well known in the art and include any suitable culturing conditions disclosed herein. Thus in certain embodiment, the methods of the present disclosure further include a step of culturing the host cell under conditions sufficient to ferment the cellodextrin. In some embodiments, the fermentation of the cellodextrin results in the production of a fermentation product. In other embodiments, the fermentation product is a fuel. In certain preferred embodiments, the fuel is ethanol or butanol. Preferably, the fuel is ethanol. In some embodiments, the ethanol is produced with an ethanol productivity that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5- fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the ethanol productivity of ethanol produced by a corresponding cell lacking the mutant cellodextrin transporter

Methods of Increasing Growth of a Cell

[0146] The present disclosure further provides methods of increasing the growth of a host cell. In one aspect, the present disclosure provides methods of increasing growth of a cell, including a first step of providing a host cell of the present disclosure containing a recombinant polynucleotide encoding a mutant cellodextrin transporter, and a second step of culturing the host cell in a medium containing cellodextrin, where the host cell grows at a faster rate in the medium than a cell that does not contain the mutant cellodextrin transporter. Any host cell of the present disclosure as described in the section entitled "Host Cells with Increased Cellodextrin Transport" may be used. The growth rate of a host cell may be measured by any method known in the art. Typically, growth rate of a cell will be measured by evaluating cell concentration in suspension by optical density. Preferably, the host cell containing the recombinant

polynucleotide and the host cell that does not contain the recombinant polynucleotide will otherwise be identical in genetic background. Media containing cellodextrins may have resulted from enzymatic treatment of biomass polymers such as cellulose.

[0147] In some embodiments, the rate of growth of the host cell containing a recombinant polynucleotide encoding a mutant cellodextrin transporter is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25 fold, at least 2.5-fold, at least 2.75-fold, at least 3-fold, at least 3.25 fold, at least 3.5-fold, at least 3.75-fold, at least 4-fold, at least 4.25 fold, at least 4.5-fold, at least 4.75-fold, at least 5-fold, at least 5.25 fold, at least 5.5-fold, at least 5.75-fold, at least 6-fold, or more higher than the rate of growth of a corresponding cell lacking the mutant cellodextrin transporter.

[0148] In methods of increasing growth of a host cell, the culturing medium may contain a cellulase-containing enzyme mixture from an altered organism, where the mixture has reduced β- glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. The organism may be altered to reduce the expression of β-glucosidase, such as by mutation of a gene encoding β-glucosidase or by targeted RNA interference or the like.

[0149] In certain embodiments, the host cell also contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. The one or more enzymes may be, for example, L-arabinose isomerase, L- ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L- arabitinol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, or any other pentose utilization enzymes known in the art.

[0150] In another aspect, the present disclosure provides methods of increasing growth of a host cell on a biomass polymer. In preferred embodiments, the biomass polymer is cellulose. In other preferred embodiments, the biomass polymer is hemicellulose. According to one aspect of the present disclosure, the method includes providing a host cell of the present disclosure containing a recombinant polynucleotide encoding a mutant cellodextrin transporter. According to yet another aspect of the present disclosure, the method includes culturing the host cell in a medium containing the biomass polymer wherein the host cell grows at a faster rate in the medium than a cell that does not contain the recombinant polynucleotide.

[0151] Methods of the present disclosure may include culturing the host cell such that recombinant polynucleotide in the cell is expressed. For microbial hosts, this process entails culturing the cells in a suitable medium. Typically cells are grown at 35°C in appropriate media. Preferred growth media in the present disclosure include, for example, common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular host cell will be known by someone skilled in the art of microbiology or fermentation science. Temperature ranges and other conditions suitable for growth are known in the art (see, e.g., Bailey and Ollis 1986).

[0152] The source of the biomass polymer in the medium may include, for example, grasses (e.g., switchgrass, Miscanthus), rice hulls, bagasse, cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, leaves, grass clippings, corn stover, corn cobs, distillers grains, legume plants, sorghum, sugar cane, sugar beet pulp, wood chips, sawdust, and biomass crops (e.g., Crambe). In addition to a biomass polymer, the medium must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures. The rate of growth of the host cell may be measured by any methods known to one of skill in the art.

[0153] In certain embodiments of the present disclosure, the expression of cellulases is increased in the host cell upon expression of a recombinant polynucleotide. "Cellulase" as used herein refers to a category of enzymes capable of hydrolyzing cellulose polymers to shorter cello-oligosaccharide oligomers, cellobiose, and/or glucose. Cellulases include, without limitation, exoglucanases, exocellobiohydrolases, endoglucanases, and glucosidases. Expression of cellulases may be measured by RT-PCR or other methods known in the art.

[0154] In certain embodiments of the present disclosure, the expression of hemicellulases is increased in the host cell upon expression of a recombinant polynucleotide. "Hemicellulase" as used herein refers to a category of enzymes capable of hydrolyzing hemicellulose polymers. Hemicellulases include, without limitation, xylanases, mannanases, arabinases (both endo and exo kinds) and their corresponding glycosidases. Expression of hemicellulases may be measured by RT-PCR or other methods known in the art.

Methods of Co-Fermentation

[0155] Another aspect of the present disclosure provides methods of co-fermenting cellulose-derived and hemicellulose-derived sugars. As used herein, "co-fermentation" refers to simultaneous utilization by a host cell of more than one sugar in the same vessel. The method includes the steps of providing a host cell, where the host cell contains a first recombinant polynucleotide encoding a mutant cellodextrin transporter of the present disclosure, and a second recombinant polynucleotide encoding a catalytic domain of a β-glucosidase and/or of a cellobiose phosphorylase of the present disclosure, and culturing the host cell in a medium containing a cellulose-derived sugar and a hemicellulose-derived sugar, where expression of the recombinant polynucleotides enables co-fermentation of the cellulose-derived sugar and the hemicellulose-derived sugar. Any host cell of the present disclosure as described in the section entitled "Host Cells with Increased Cellodextrin Transport" may be used.

[0156] In certain embodiments, the host cell also contains one or more recombinant polynucleotides where the one or more polynucleotides encode one or more enzymes involved in pentose utilization. Alternatively, one or more polynucleotides encoding one or more enzymes involved in pentose utilization may be endogenous to the host cell. The one or more enzymes may include, for example, L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabitinol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, or any other pentose-utilizing enzymes known to one of skill in the art.

[0157] In certain embodiments, the host cell contains a third recombinant polynucleotide where the polynucleotide encodes a pentose transporter. Alternatively, the host cell may contain an endogenous polynucleotide encoding a pentose transporter. In preferred embodiments, the pentose transporter transports xylose and/or arabinose into the cell. In certain embodiments, the third recombinant polynucleotide encodes a polypeptide such as NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUT1, or XUT3. The expression of a pentose transporter in the host cell may enhance the efficiency of co-fermentation if glucose is present along with a pentose sugar is the growth medium.

[0158] In methods of co-fermentation as described herein, cellulose-derived sugars preferably include cellobiose, cellotriose, and celltetraose, and hemicellulose-derived sugars preferably include xylose and arabinose. Typically, in order to prepare the cellulose-derived sugars and hemicellulose-derived sugars for co-fermentation by a host cell, lignocellulosic biomass is first pretreated to alter its structure and allow for better enzymatic hydrolysis of cellulose. Pretreatment may include physical or chemical methods, including, for example, ammonia fiber/freeze explosion, the lime method based on calcium or sodium hydroxide, and steam explosion with or without an acid catalyst. Acid treatment will release xylose and arabinose from the hemicellulose component of the lignocellulosic biomass. Next, preferably, the cellulose component of the pretreated biomass is hydrolyzed by a mixture of cellulases. Examples of commercially available cellulase mixtures include Celluclast 1.5L^® (Novozymes), Spezyme CP^® (Genencor) (Scott W. Pryor, 2010, Appl Biochem Biotechnol), and Cellulyve 50L (Lyven).

[0159] Cellulase mixtures typically contain endoglucanases, exoglucanases, and β- glucosidases. In methods of co-fermentation as described herein, the amount of β-glucosidase activity in the cellulase mixture should be minimized as much as possible. For example, the culturing medium may contain a cellulase-containing enzyme mixture from an altered organism, where the mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism. The organism may be altered to reduce the expression of β- glucosidase, such as by mutation of a gene encoding β-glucosidase or by targeted RNA interference or the like.

Methods of Synthesis of Hydrocarbons or Hydrocarbon Derivatives

[0160] Another aspect of the present disclosure provides methods for increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell of the present disclosure containing a recombinant polynucleotide encoding a mutant cellodextrin transporter.

[0161] "Hydrocarbons" as used herein are organic compounds consisting entirely of hydrogen and carbon. Hydrocarbons include, without limitation, methane, ethane, ethene, ethyne, propane, propene, propyne, cyclopropane, allene, butane, isobutene, butene, butyne, cyclobutane, methylcyclopropane, butadiene, pentane, isopentane, neopentane, pentene, pentyne, cyclopentane, methylcyclobutane, ethylcyclopropane, pentadiene, isoprene, hexane, hexene, hexyne, cyclohexane, methylcyclopentane, ethylcyclobutane, propylcyclopropane, hexadiene, heptane, heptene, heptyne, cycloheptane, methylcyclohexane. heptadiene, octane, octene, octyne, cyclooctane, octadiene, nonane, nonene, nonyne, cyclononane, nonadiene, decane, decene, decyne, cyclodecane, and decadiene. [0162] "Hydrocarbon derivatives" as used herein are organic compounds of carbon and at least one other element that is not hydrogen. Hydrocarbon derivatives include, without limitation, alcohols (e.g. , arabinitol, butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, and xylitol); organic acids (e.g. , acetic acid, adipic acid, ascorbic acid, citric acid, 2,5- diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, and xylonic acid); esters; ketones (e.g. , acetone); aldehydes (e.g. , furfural); amino acids (e.g. , aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); and gases (e.g. , carbon dioxide and carbon monoxide).

[0163] In preferred embodiments, the hydrocarbon or hydrocarbon derivative can be used as fuel. In particularly preferred embodiments, the hydrocarbon or hydrocarbon derivative is ethanol or butanol.

[0164] According to one aspect of the present disclosure, a method of increasing the synthesis of hydrocarbons or hydrocarbon derivatives by a host cell includes a first step of providing a host cell, where the host cell contains a recombinant polynucleotide encoding a mutant cellodextrin transporter of the present disclosure, and a second step of culturing the host cell in a medium containing cellodextrin or a source of cellodextrin to increase the synthesis of hydrocarbons or hydrocarbon derivatives by the host cell, where transport of cellodextrin into the cell is increased upon expression of the recombinant polynucleotide compared to a

corresponding cell lacking the mutant cellodextrin transporter. Any host cell of the present disclosure as described in the section entitled "Host Cells with Increased Cellodextrin Transport" may be used. Preferably, the host cell containing the recombinant polynucleotide and the host cell that does not contain the recombinant polynucleotide will otherwise be identical in genetic background.

[0165] The culturing medium may contain a cellulase-containing enzyme mixture from an altered organism, where the mixture has reduced β-glucosidase activity compared to a cellulase- containing mixture from an unaltered organism. The organism may be altered to reduce the expression of β-glucosidase, such as by mutation of a gene encoding β-glucosidase or by targeted RNA interference or the like. [0166] It is to be understood that, while the present disclosure has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure pertains.

[0167] The following examples are offered to illustrate provided embodiments and are not intended to limit the scope of the present disclosure.

EXAMPLE

[0168] The following example relates to the identification and characterization of the Scheffersomyces stipitis HXT2.4 protein as a cellobiose transporter; the identification and characterization of a mutant CEN-HXT2.4-BGL strain with improved cellobiose fermentation activity; and the characterization of mutant HXT2.4 transporters containing negatively charged amino acid substitutions at position 291.

Materials and Methods

Strains and plasmid constructions

[0169] S. cerevisiae CEN. PK2-1D (MATalpha, leu2, trpl, ura3, his3, MAL2-8^C, SUC2) and S. cerevisiae D452-2 (MATalpha, leu2, his3, ura3, and canl) were used for engineering of cellobiose metabolism in yeast. Escherichia coli DH5 (F- recAl endAl hsdR17 [rK- mK+] supE44 thi-1 gyrA relAl) (Invitrogen, Gaithersburg, MD) was used for gene cloning and manipulation. In order to overexpress HXT2.4 in S. cerevisiae, the pRS426 plasmid was used. PGK promoter and CYC1 terminator were used to overexpress HXT2.4 as we constructed pRS426-cdt-l for overexpressing cdt-1 previously (6). pRS425-ghl-l containing ghl-1 under the control of PGK promoter and CYC1 terminator was co-expressed to enable intracellular utilization of cellobiose (6). Medium and culture conditions

[0170] E. coli was grown in Luria-Bertani medium; 50 μg/ml of ampicillin was added to the medium when required. Yeast strains were routinely cultivated at 30°C in YP medium (10 g/L yeast extract and 20 g/L Bacto peptone) with 20 g/L of glucose. To select transformants using an amino acid auxotrophic marker, yeast synthetic complete (YSC) medium was used, which contained 6.7 g/L of yeast nitrogen base plus 20 g/L of glucose, 20 g/L of agar, and CSM-Leu- Trp-Ura (Bio 101, Vista, CA) which supplied appropriate nucleotides and amino acids.

Yeast transformation and plasmid isolation

[0171] Transformation of expression cassettes for constructing xylose and cellobiose metabolic pathways was performed using the yeast EZ-Transformation kit (BIO 101, Vista, CA). Transformants were selected on YSC medium containing 20 g/L of glucose or cellobiose. Amino acids and nucleotides were added as necessary. Plasmid isolation from an evolved S. cerevisiae was performed using Zymoprep™ Yeast Plasmid Miniprep I kit (Zymo Research Inc., Orange, CA) according to the supplier's instructions.

Saturation mutagenesis

[0172] Saturation mutagenesis was carried out using the Quik-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA) to generate all possible replacements at the A291 residue of HXT2.4. A library of random mutant HXT2.4 (A291X) was synthesized with a set of degenerate mutagenic primers using pRS426-H¾ 2.4 as a template. PCR was performed on a CI 000™ thermal cycler (Bio-Rad, USA) under the following conditions: an initial denaturation step for 30 sec at 98°C followed by 16 repeating cycles of 20 sec at 98°C, 30 sec at 50°C, 5 min at 72°C, and a final step of 10 min at 72°C. The following primers were used:

HXT_A291X-1: 5 '-GAAAAGTTATATNNNAGCTCTTCTTAC-3 ' (SEQ ID NO: 28)

HXT_A291X-2: 5 '-GTA AGAAGAGCTNNNATATAACTTTTC-3 ' (SEQ ID NO: 29) Fermentation experiments

[0173] Yeast cells were grown in YP medium containing 20 g/L of cellobiose to prepare inoculums for cellobiose fermentation experiments. Cells at mid-exponential phase from YP medium containing cellobiose were harvested and inoculated after washing twice with sterilized water. All flask fermentation experiments were performed using 50 mL of YP medium containing 80 g/L of cellobiose in 250 mL flask at 30°C with initial Οϋ₆οο of ~ 1.0 under oxygen limited conditions.

[0174] Laboratory evolution of an HXT2.4 expressing strain was performed by serial-sub cultures on cellobiose. When cellobiose concentrations reached to almost zero, cells were collected and used to established new reactions at an Οϋ₆οο of -0.01. This process was repeated 9 times over the course of 50 days. Then cells were plated onto minimal medium agar plates containing 20 g/L of cellobiose to isolate a single colony. After the confirmation of improved phenotypes from the isolated single colony, a plasmid was isolated and sequenced.

[0175] Small scale fermentations were also performed in 200

of YP medium containing 5 g/L of cellobiose, cellotriose, or cellotetraose using a Biotek Synergy HT spectrophotometer (Biotek, Winooski, VT) with a 96 well plate. A culture volume in each well was 200 \L and 50\L of mineral oil was overlaid on the top of the culture to prevent evaporation of the medium during growth measurement. The initial cell density was adjusted to Οϋ₆οο = -0.1. Synergy H4 hybrid Microplate Reader (BioTek Instruments Inc., Winooski, VT) was used for measuring absorbance at 600 nm with a continuous mixing option. Cellotriose and cellotetraose was obtained from Seikagaku Biobusiness Corporation.

Measurement of GFP fluorescence

[0176] During fermentation, when OD (600 nm) reached 10.0, cells were harvested and washed twice with sterilized water. A 200 μΐ cell suspension was then transferred to a black 96- well optical bottom plate (Corning, NY). Fluorescence intensities were measured with a Biotek Synergy HT spectrophotometer (Biotek, Winooski, VT) at an excitation wavelength of 485 nm, emission of 528 nm. H] -Cellobiose Transport Assays and Kinetic Parameters

[0177] Transport assays were performed using a modification of the oil- stop method (2). Yeast recombinant strains expressing transporter genes fused to GFP were grown to the mid- exponential phases in selective media, washed 3x with assay buffer (30 mM MES-NaOH [pH 5.6] and 50 mM ethanol), and resuspended to an OD (600 nm) of 40. To start transport reactions, 50 μΐ_^ of cells were added to 50 μΐ_^ of [ H] -cellobiose layered over 100 μΐ_^ of silicone oil (Sigma 85419). Reactions were stopped by spinning cells through oil for 1 minute at 17,000 g, tubes were frozen in ethanol/dry ice, and tube-bottoms containing the cell-pellets were clipped off into 1 mL of 0.5 M NaOH. The pellets were solubilized overnight, 5 mL of Ultima Gold scintillation fluid added, and CPM determined in a Tri-Carb 2900TR scintillation counter. [ H] -cellobiose was purchased from Moravek Biochemicals, Inc., and had a specific activity of 4 Ci/mmol and a purity of >99 .

[0178] Kinetic parameters were determined by measuring the linear rate of [³H] -cellobiose uptake over 3 minutes for cellobiose concentrations between 0.5 and 400 μΜ. V_max and K_m values were determined by fitting a single rectangular, 2-parameter hyperbolic function to a plot of rates vs. cellobiose concentration by non-linear regression in SigmaPlot^®. V_max values were normalized for differences in transporter abundance by measuring the GFP fluorescence from 200 μΐ_^ of cells at OD (600 nm) 40 immediately before beginning transport assays. Kinetic parameters reported in the text are the mean + SEM from three separate experiments.

Analytical methods

[0179] Cell growth was monitored by optical density (OD) at 600 nm using UV- visible Spectrophotometer (Biomate 5, Thermo, NY). Glucose, xylose, xylitol, glycerol, acetate and ethanol concentrations were determined by high performance liquid chromatography (HPLC, Agilent Technologies 1200 Series) equipped with a refractive index detector using a Rezex ROA-Organic Acid H+ (8%) column (Phenomenex Inc., Torrance, CA). The column was eluted with 0.005 N of H₂S0₄ at a flow rate of 0.6 ml/min at 50°C. Results

Expression ofS. stipitis HXT2.4 from in S. cerevisiae with intracellular β-glucosidase

[0180] Schejfersomyces stipitis can ferment cellobiose and produce ethanol with

approximately 0.30 to 0.41 g/g of yield and approximately 0.15 to 0.21 g/L-h of volumetric productivity. Through a BLAST search using the amino acid sequence of the N. crassa cellodextrin transporter CDT-1, several transporters having high homology with CDT-1 were identified from the genome sequence of S. stipitis. The identified homologs included putative hexose transporters (HXT2.1, HXT2.3, HXT2.4, HXT2.5 and HXT2.6), and lactose permeases (LACl, LAC2, and LAC3). Among them, HXT2.4, which has 31% sequence identity with CDT-1, was found to be located near the endo-l,4-P-glucanase (EGC2) and β-glucosidase (BGL5) genes in chromosome 1 of S. stipitis (Fig. 1). This co-location indicated that HXT2.4 is a putative cellodextrin transporter.

[0181] Homolog search to identify putative cellobiose transporters in Schejfersomyces stipitis was performed using amino acid sequence of CDT-1 or CDT-2 from N. crassa. Proteins annotated as lactose transporters (LACl, LAC2, and LAC3), and putative hexose transporters (HXT2.1, HXT2.3, HXT2.4, HXT2.5, and HXT2.6) were found to have high sequence identities with CDT-1 (29-32%) and CDT-2 (29-36%). Among the identified proteins, HXT2.4, which has 31% sequence identity with CDT-1 and 36% sequence identity with CDT-2, was found to be located near the endo-l,4-P-glucanase (EGC2) and β-glucosidase (BGL5) genes in chromosome 1 of S. stipitis (Fig. 1). This co-location indicated that HXT2.4 is a putative cellodextrin transporter.

[0182] HXT2.4 was cloned and ligated with pRS426 vector under the control of PGR promoter and CYC1 terminator same as pRS426-c¾¾-i described in previous paper (6). After overexpressing HXT2.4 or cdt-1 with ghl-1 in S. cerevisiae (CEN. PK2-1D), cellobiose fermentation rates by the resulting strains (CEN-HXT2.4-BGL or CEN-CDT1-BGL) were investigated (Fig. 2). The CEN-HXT2.4-BGL strain consumed 72 g/L of cellobiose and produced 29 g/L of ethanol in 54 h, whereas the CEN-CDT1-BGL strain consumed 79 g/L of cellobiose and produced 31 g/L of ethanol in 36 h. Although the ethanol yield from the CEN- HXT2.4-BGL strain (0.40 g/g) was similar to that from the CEN-CDT 1 -BGL strain( 0.39 g/g), the cellobiose consumption rate (1.33 g/L^»h) and ethanol productivity (0.54 g/L^»h) of the CEN- HXT2.4-BGL strain were much lower than that of the CEN-CDT 1 -BGL strain (2.19 g/L^«h and 0.86 g/L'h) (Fig. 2).

Isolation of an HXT2.4 mutant with faster cellobiose fermentation rate

[0183] In order to improve the cellobiose fermentation rate of the CEN-HXT2.4-BGL strain, an evolutionary engineering approach, based on serial sub-cultures of the strain on cellobiose, was performed. Specific growth rates of the strains growing on YP medium containing 80 g/L of cellobiose increased drastically, from 0.029 h^"1 to 0.080 h^"1, during the serial transfers (Fig. 3). After the ninth serial sub-culture, where the specific growth rate of the culture did shown any further improvement, the evolved culture was plated on a cellobiose containing plate to isolate a single colony. The cellobiose fermentation rate by the single colony (evolved CEN-HXT2.4- BGL) was examined using YP medium containing 80 g/L of cellobiose. The evolved CEN- HXT2.4-BGL strain consumed 75 g/L of cellobiose and produced 32 g/L of ethanol within 36 h, resulting in 2.08 g/L^»h of cellobiose consumption rate, 0.43 g/g of ethanol yield, and 0.88 g/L^»h of ethanol productivity (Fig. 4).

[0184] As compared to its parental strain (CEN-HXT2.4-BGL), the evolved strain showed much faster cellobiose fermentation. The cellobiose consumption rate increased from 1.33 g/L^»h to 2.08 g/L-h (56% improvement), and ethanol productivity increased from 0.54 g/L^»h to 0.83 g/L^»h (53% improvement) (Figs. 4A and 4C). As a result, cell growth, cellobiose consumption, and ethanol production by the evolved CEN-HXT2.4-BGL strain were almost comparable with those by the CEN-CDT 1 -BGL strain.

[0185] Moreover, the amount of accumulated cellodextrin (e.g., cellotriose and cellotetraose) by the evolved CEN-HXT2.4-BGL strain was lower than that of the CEN-CDT 1 -BGL strain, resulting in a higher ethanol yield (0.43 g/g vs. 0.39 g/g) (Fig. 4D).

Identification of a single amino acid substitution in HXT2.4 of evolved strain

[0186] In order to identify the genetic changes responsible for the improved cellobiose fermentation by the evolved CEN-HXT2.4-BGL strain, we isolated and sequenced two plasmids (pRS426-HXr2.4 and pRS425-ghl-l) containing the HXT2.4 and ghl-1 genes from the evolved strain. Analysis of the pRS425-ghl-l revealed that the plasmid contained no mutations.

However, analysis of pRS426-HXT2.4 plasmid revealed that the HXT2.4 coding region contained a single nucleotide mutation (C872A). This mutation results in an amino acid substitution from alanine (A) to aspartate (D) at position 291 (A291D) of the translated HXT2.4 polypeptide.

[0187] The location of the A291D mutation in HXT2.4 was predicted by a protein structure prediction method (Phyre2) and modeled by I-TASSER using the E. coli lactose permease crystal structure (11). According to the prediction, A291D is located in the cytoplasmic regions between the 6th and 7th membrane-spanning loops of HXT2.4 (Figs. 5 and 6).

[0188] No mutations were found in any other regions of the pRS426-H¾ 2.4 plasmid, such as the promoter, origin of replication, or auxotrophic markers of the isolated plasmids from the evolved strains.

[0189] We also confirmed the beneficial effect of the A291D mutation in ΗΧΤ2.4 by re- transforming into the parental strain both the isolated plasmid a synthesized plasmid containing the A291D mutation generated by site-directed mutagenesis. The resulting strains showed almost identical cellobiose fermentation rates as compared to the evolved CEN-HXT2.4-BGL strain (data not shown), suggesting that there were no chromosomal mutations contributing to the improved cellobiose fermentation rate phenotype of the evolved strain.

[0190] The amino acid sequence of the mutant HXT2.4 containing the A291D mutation was also aligned with the amino acid sequence of the N. crassa transporter CDT-1 (Fig. 7). The alignment shows that the amino acid residue on CDT-1 that corresponds to A291 is K308.

Substitution of alanine with negatively charged amino acids (aspartate and glutamate) at the position 291 ofHXT2.4

[0191] In order to investigate the effect on cellobiose fermentation of various amino acid substitutions at position 291, we performed saturation mutagenesis experiments. A library of random mutants (pRS426-H¾ 2.4-A291X) was constructed using a Quick Change site-directed mutagenesis kit with degenerated primers (12). The diversity of the mutant library was confirmed by sequencing the isolated plasmids from ten randomly picked colonies. We found that the ten sequenced plasmids each contained different nucleotides (GAA, GGG, CAT, AAA, TTC, TTA, CTA, TCT, TCA, and TAT) at positions 871-873, which result in amino acid substitutions from alanine to glutamate, glycine, histidine, lysine, leucine, serine, and tyrosine at position 291 of the HXT2.4 polypeptide. After introducing each plasmid, which contained an HXT2.4 mutant along with pRS425-ghl-l, into S. cerevisiae, the cellobiose consumption rate and ethanol production rate of each transformant was compared to that of a control strain containing the wild type HXT2.4 (Table 2 and Fig. 8).

Table 2

Nucleotide Amino acid Cellobiose Ethanol

at position At position consumption rate production rate

871-873 291 (g L-h) (g L-h)

GCC Alanine (WT) 0.94 0.15

GAA Glutamate 3.18 1.16

AAA Lysine 2.14 0.88

CTA Leucine 1.55 0.41

TTA Leucine 1.09 0.20

TTG Leucine 0.77 0.14

TAT Tyrosine 0.91 0.15

TCT Serine 0.53 0.06

TCA Serine 0.51 0.05

GGG Glycine 0.42 0.01

CAT Histidine No growth N/A

[0192] As expected, all transformants showed varied cellobiose fermentation rates that depended on the particular mutation at position 291 of the Hxt2.4 polypeptide. The replacement of alanine to glutamate (A291E) resulted in the fastest cell growth, cellobiose consumption, and ethanol production among the transformants. The transformant with the A291E mutation consumed 78 g/L of cellobiose within 24 h, while the wild type HXT2.4 transformant (D452- HXT2.4 -BGL) consumed only 56 g/L of cellobiose in 60 h. However, the replacement of alanine to serine or glycine (A291S or A291G) resulted in slower cellobiose consumption compared to that of the wild type HXT2.4. Interestingly, when alanine was changed to histidine (A291H), the transformant was not able to grow and consume cellobiose at all.

[0193] In order to isolate HXT2.4 mutants capable of facilitating cellobiose fermentation that is faster than the A291D mutation, fast growing transformants on cellobiose containing plates were isolated after transforming the random library of mutant HXT2.4 (A291X) with pRS425- ghl-1 into S. cerevisiae. Nineteen fast- growing colonies on minimal medium agar plates containing 20 g/L of cellobiose were isolated. As expected, all isolated transformants showed faster cellobiose fermentation rates as compared to a control strain (Fig. 14). We also isolated and sequenced plasmids from the nineteen transformants. Sequence analysis of the isolated plasmids revealed that replacements of alanine to glutamate or aspartate (A291E or A291D) facilitated the fastest cell growth, cellobiose consumption, and ethanol production among the nineteen transformants (Fig. 14).

[0194] In order to contextualize the effect on cellobiose fermentation of amino acid substitution at position 291 of the HXT2.4 polypeptide (A291X), we compiled all fermentation results (Fig. 9). Amino acid substitutions from alanine to negatively charged amino acids (glutamate or aspartate) resulted in the highest cellobiose fermentation capability. Additionally, replacement of alanine with positively charged amino acids (lysine or arginine) resulted in improved cellobiose fermentation, although they were slightly less effective than the negatively charged amino acids. Amino acid substitutions from alanine to valine or leucine resulted in slight improvements on cellobiose fermentation. Interestingly, the improved cellobiose fermentation rates from amino acid substitutions were inversely proportional to the maximum amount of accumulated cellodextrin (Figs. 9 and 10). Cellobiose fermentation profiles by representative transformants containing various amino acid substitutions clearly showed that A291 is a key amino acid residue affecting cellobiose fermentation rates by the engineered S. cerevisiae (Fig. 11).

Kinetic studies of mutant HXT2.4

[0195] It was thought that the amino acid substitution at position 291 of the mutant HXT2.4 polypeptide may cause better protein folding or localization in the plasma membrane as well as higher cellobiose transport capability, which leads to fast cellobiose fermentation. In order to compare protein folding or localization between the wild type HXT2.4 and the mutant HXT2.4 (A291D), we overexpressed green fluorescent protein (GFP) tagged HXT2.4-GFP or HXT2.4 (A291D)-GFP in S. cerevisiae with ghl-1. When the ODs reached -10.0 during cellobiose fermentation, cells were harvested and GFP fluorescence was measured after washing twice with sterilized water. The GFP fluorescence levels of both HXT2.4-GFP and HXT2.4 (A291D)-GFP were similar (15668+155 and 16559+147), and twenty-fold higher than that from HXT2.4 or HXT2.4 (A291D) without the GFP tag (-800). Kinetic properties of wild type HXT2.4 and mutant HXT2.4 (A291D) were determined by measuring the rate of [ H]-cellobiose uptake into cells (Fig. 12 and Table 3).

Table 3

[0196] Table 3 shows transport kinetics of wild type HXT2.4 and mutant HXT2.4 (A291D). The kinetic parameters were determined via non-linear regression from the datasets in Figure 12. All values were normalized to 10 million GFP fluorescence.

[0197] Mutant HXT2.4 (A291D) exhibited four-fold higher maximal cellobiose transport rates (V_max) and 1.5-fold higher K_m as compared to wild type HXT2.4 (Table 3). Based on these results, it is believed that the improved cellobiose fermentation rate of the A291D mutant was due to different kinetic properties of the mutant polypeptide, and not to improved protein folding or localization to the plasma membrane. As such, it is believed that charged amino acids located in the cytoplasmic region of the HXT2.4 polypeptide may enhance cellobiose transport capability, as replacement of alanine with negatively charged amino acids or positively charged amino acids at position 291 of HXT2.4 achieved the highest cellobiose fermentation capability and the location of amino acid 291 is predicted to be in the cytoplasmic region between the sixth and seventh membrane-spanning loops (Figs. 5 and 6). This is similar to the. E. coli melibiose (D-Gal-a(l→6)-D-Glc) permease (MelB), which is a symporter that can be inactivated by the replacement of a positively charged amino acid (arginine) with neutral amino acids (cysteine or glutamine) (1).

[0198] In order to determine cellodextrin transport capabilities of wild type HXT2.4 and mutant HXT2.4 (A291D), growth assays were performed in YP medium containing cellobiose, cellotriose, or cellotetraose. CDT-1 was used as a positive control because CDT-1 has cellodextrin transport capability (4). With cellobiose, CDT-1 and mutant HXT2.4 (A291D) transformants grew well, while the wild type HXT2.4 transformant showed poor growth as we expected (Fig. 13A). Interestingly, mutant HXT2.4 (A291D) transformant showed even better cell growth than the CDT- 1 or wild type HXT2.4 transformant with cellotriose or cellotetraose (Figs. 13B and 13C). These results suggest that amino acids substitutions at position 291 of HXT2.4 affect not only cellobiose transport capability, but also the capability of transporting higher degree of polymerization (DP) cellodextrins (e.g., cellotriose and cellotetraose).

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Claims

CLAIMS We claim:

1. A host cell comprising, a recombinant polynucleotide encoding a mutant cellodextrin transporter, wherein the mutant cellodextrin transporter comprises a transmembrane a- helix 1, a-helix 2, a-helix 3, a-helix 4, a-helix 5, a-helix 6, a-helix 7, a-helix 8, a-helix 9, a-helix 10, a-helix 11, a-helix 12, and a loop sequence positioned between a-helix 6 and a-helix 7, wherein the mutant cellodextrin transporter comprises at least one mutation in the loop sequence, and wherein the mutant cellodextrin transporter has a V„ that is at least 1-fold higher than the V_», of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence.

2. The host cell of claim 1, wherein the at least one mutation is an amino acid substitution.

3. The host cell of claim 1 or claim 2, wherein the loop sequence corresponds to amino acids 237-306 of SEQ ID NO: 1.

4. The host cell of claim 3, wherein the at least one mutation is an amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1.

5. The host cell of claim 4, wherein the at least one mutation is selected from the group consisting of an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Arg amino acid substitution at a position

corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Lys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Glu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Gin amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Cys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Phe amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Leu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Asn amino acid

substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Thr amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, an Ala to Val amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1, and combinations thereof.

6. The host cell of claim 4, wherein the at least one mutation is an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1.

7. The host cell of claim 4, wherein the at least one mutation is an Ala to Arg amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1.

8. The host cell of claim 4, wherein the at least one mutation is an Ala to Lys amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1.

9. The host cell of claim 4, wherein the at least one mutation is an Ala to Glu amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1.

10. The host cell of claim 4, wherein the at least one mutation is an Ala to Gin amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 1.

11. The host cell of any one of claims 1-10, wherein transmembrane a-helix 1 comprises SEQ ID NO: 3.

12. The host cell of any one of claims 1-11, wherein transmembrane a-helix 2 comprises SEQ ID NO: 4.

13. The host cell of any one of claims 1-12, wherein the mutant cellodextrin transporter further comprises a loop connecting transmembrane a-helix 2 and transmembrane a-helix 3 comprising SEQ ID NO: 5.

14. The host cell of any one of claims 1-13, wherein transmembrane a-helix 5 comprises SEQ ID NO: 6.

15. The host cell of any one of claims 1-14, wherein transmembrane a-helix 6 comprises SEQ ID NO: 7.

16. The host cell of any one of claims 1-15, wherein the mutant cellodextrin transporter further comprises a sequence between transmembrane a-helix 6 and transmembrane a- helix 7 comprising SEQ ID NO: 8.

17. The host cell of any one of claims 1-16, wherein transmembrane a-helix 7 comprises SEQ ID NO: 9.

18. The host cell of any one of claims 1-17, wherein transmembrane a-helix 10,

transmembrane a-helix 11, and the sequence between a-helix 10 and a-helix 11 comprise SEQ ID NO: 10.

19. The host cell of any one of claims 1-18, wherein the mutant cellodextrin transporter further comprises a loop connecting transmembrane a-helix 2 and transmembrane a-helix 3 comprising SEQ ID NO: 5, transmembrane a-helix 5 comprises SEQ ID NO: 6, transmembrane a-helix 6 comprises SEQ ID NO: 7, the mutant cellodextrin transporter further comprises a sequence between transmembrane a-helix 6 and transmembrane a-helix 7 comprising SEQ ID NO: 8, and transmembrane a-helix 10, transmembrane a-helix 11, and the sequence between a-helix 10 and a-helix 11 comprise SEQ ID

NO: 10.

20. The host cell of any one of claims 1-19, wherein the mutant cellodextrin transporter comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to SEQ ID NO: 2.

21. The host cell of any one of claims 1-20, wherein the V„ of the mutant cellodextrin transporter is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5- fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, or at least 6-fold higher than the V_», of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence.

22. The host cell of any one of claims 1-21, wherein the mutant cellodextrin transporter has a K_m that is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5- fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold higher than the K_m of a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence.

23. The host cell of any one of claims 1-22, wherein the host cell exhibits a cellodextrin

consumption rate that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35- fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6- fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85- fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, or at least 3-fold higher than the cellodextrin consumption rate exhibited by a corresponding cell lacking the mutant cellodextrin transporter.

24. The host cell of any one of claims 1-23, wherein the host cell exhibits an ethanol

productivity that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4-fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65-fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9-fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, or at least 3-fold higher than the ethanol productivity exhibited by a corresponding cell lacking the mutant cellodextrin transporter.

25. The host cell of any one of claims 1-24, wherein the host cell further comprises a

recombinant polynucleotide encoding at least a catalytic domain of a cellodextrin phosphorylase.

26. The host cell of claim 25, wherein the cellodextrin phosphorylase comprises an amino acid sequence that has at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 20 (CDP_Clent), SEQ ID NO: 21 (CDP_Ctherm), and SEQ ID NO: 22

(CDP_Acell).

27. The host cell of claim 25, wherein the cellodextrin phosphorylase has cellobiose

phosphorylase activity.

28. The host cell of claim 27, wherein the cellodextrin phosphorylase with cellobiose phosphorylase activity comprises an amino acid sequence that has at least 90%, at least 95%, at least 99%, or at least 100% amino acid identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 23 (CgCBP), SEQ ID NO: 24 (SdCBP), and SEQ ID NO: 25 (CtCBP).

29. The host cell of any one of claims 1-28, wherein the host cell further comprises a

recombinant polynucleotide encoding at least a catalytic domain of a β-glucosidase.

30. The host cell of claim 29, wherein the β-glucosidase is from Neurospora crassa.

31. The host cell of claim 29, wherein the β-glucosidase is encoded by NCU00130.

32. The host cell of any one of claims 1-31, wherein the host cell further comprises one or more recombinant polynucleotides encoding one or more enzymes involved in pentose utilization.

33. The host cell of claim 32, wherein the one or more enzymes are selected from one or more of the group consisting of L-arabinose isomerase, L-ribulokinase, L-ribulose-5-P 4 epimerase, xylose isomerase, xylulokinase, aldose reductase, L-arabinitol 4- dehydrogenase, L-xylulose reductase, and xylitol dehydrogenase.

34. The host cell of any one of claims 1-33, wherein the host cell further comprises a

recombinant polynucleotide encoding a pentose transporter.

35. The host cell of claim 34, wherein the pentose transporter is selected from the group consisting of NCU00821, NCU04963, NCU06138, STL12/XUT6, SUT2, SUT3, XUTl, and XUT3.

36. The host cell of any one of claims 1-35, wherein the cellodextrin is selected from one or more of the group consisting of cellobiose, cellotriose, and cellotetraose.

37. The host cell of any one of claims 1-36, wherein the host cell is an oleaginous yeast.

38. The host cell of any one of claims 1-37, wherein the host cell is selected from the group consisting of Saccharomyces sp., Saccharomyces cerevisiae, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Saccharomyces pombe, Kluyveromyces sp., Kluyveromyces marxiamus, Kluyveromyces lactis, Kluyveromyces fragilis, Pichia stipitis, Sporotrichum thermophile, Myceliophthora thermophila, Candida shehatae, Candida tropicalis, Neurospora crassa, Zymomonas mobilis, Clostridium sp., Clostridium phytofermentans, Clostridium thermocellum, Clostridium beijerinckii, Clostridium acetobutylicum, Moorella thermoacetica,

Escherichia coli, Klebsiella oxytoca, Thermoanaerobacterium saccharolyticum, Bacillus subtilis, Rhodosporidium toruloides, Lipomyces starkyei, Yarrowia lipolytica, and Cryptococcus curvatus.

39. A method of increasing transport of cellodextrin into a cell, comprising: providing the host cell of any one of claims 1-38; and culturing the cell in a medium such that the recombinant polynucleotide is expressed, wherein expression of the recombinant polynucleotide results in increased transport of cellodextrin into the cell compared to a corresponding cell expressing a recombinant polynucleotide encoding a cellodextrin transporter lacking the at least one mutation in the loop sequence.

40. The method of claim 39, wherein the cellodextrin is transported into the cell at a that is at least 1-fold higher than the V„ of cellodextrin transport into a corresponding cell having a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence.

41. The method of claim 40, wherein the cellodextrin is transported into the cell at a V_», that is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, or at least 6-fold higher than the of cellodextrin transport into a corresponding cell having a corresponding cellodextrin transporter lacking the at least one mutation in the loop sequence.

42. The method of any one of claims 39-41, wherein the cellodextrin is consumed at a rate that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4- fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65- fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9- fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75- fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, or at least 3-fold higher than the rate of cellodextrin consumption by a corresponding cell lacking the mutant cellodextrin transporter.

43. The method of any one of claims 39-42, further comprising culturing the host cell under conditions sufficient to ferment the cellodextrin.

44. The method of claim 43, wherein the fermentation of the cellodextrin results in the

production of a fermentation product.

45. The method of claim 44, wherein the fermentation product is a fuel.

46. The method of claim 45, wherein the fuel is ethanol or butanol.

47. The method of claim 45, wherein the fuel is ethanol.

48. The method of claim 47, wherein the ethanol is produced with an ethanol productivity that is at least 0.2-fold, at least 0.25-fold, at least 0.3-fold, at least 0.35-fold, at least 0.4- fold, at least 0.45-fold, at least 0.5-fold, at least 0.55-fold, at least 0.6-fold, at least 0.65- fold, at least 0.7-fold, at least 0.75-fold, at least 0.8-fold, at least 0.85-fold, at least 0.9- fold, at least 0.95-fold, at least 1-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75- fold, at least 2-fold, at least 2.25-fold, at least 2.5-fold, at least 2.75-fold, or at least 3-fold higher than the ethanol productivity of ethanol produced by a corresponding cell lacking the mutant cellodextrin transporter.

49. The method of any of claims 39-48, wherein the medium comprises a cellulase- containing enzyme mixture from an altered organism, wherein the cellulase-containing mixture has reduced β-glucosidase activity compared to a cellulase-containing mixture from an unaltered organism.

50. An isolated polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or at least 100% identical to SEQ ID NO: 2, wherein the polypeptide comprises an Ala to Asp amino acid substitution at a position corresponding to amino acid 291 of SEQ ID NO: 2.

51. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

52. An isolated polynucleotide encoding the polypeptide of any one of claims 50 or claim 51.

53. An expression vector, comprising the isolated polynucleotide of claim 52, operably

linked to a regulatory sequence.

54. A host cell comprising the expression vector of claim 53.