# Patent

## Title
Sugar transport sequences, yeast strains having improved sugar uptake, and methods of use

## Abstract

Disclosed are nucleic acid constructs comprising coding sequences operably linked to a promoter not natively associated with the coding sequence. The coding sequences encode Pichia stipitis proteins that allow recombinant strains of Saccharomyces cerevisiae expressing the protein to grow on xylose, and allow or increase uptake of xylose by Pichia stipitis or Saccharomyces cerevisiae expressing the coding sequences. Expression of the coding sequences enhances uptake of xylose and/or glucose, allowing increased ethanol or xylitol production.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/484,714, filed Jun. 15, 2009, now U.S. Pat. No. 8,105,811, which claims priority to U.S. Provisional Patent Application No. 61/061,417 filed Jun. 13, 2008, the contents of which are incorporated by reference in their entirety.
REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The Sequence Listing written in file -23-2.TXT, created on Dec. 28, 2011, 24,576 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention is jointly owned between WARF and the USDA and was made with United States government support awarded by the following agencies:
USDA Grant Number 2006-35504-17436.
The government has certain rights in this invention.


BACKGROUND OF THE INVENTION
Within the United States, ongoing research is directed toward developing alternative energy sources to reduce our dependence on foreign oil and nonrenewable energy. The use of ethanol as a fuel has become increasingly prevalent in recent years. Currently, corn is the primary carbon source used in ethanol production. The use of corn in ethanol production is not economically sustainable and, arguably, may result in increased food costs.
In order to meet the increased demand for ethanol, it will be necessary to ferment sugars from other biomass, such as agricultural wastes, corn hulls, corncobs, cellulosic materials, pulping wastes, fast-growing hardwood species, and the like. Biomass from most of these sources contains large amounts of xylose, constituting about 20 to 25% of the total dry weight. Because agricultural residues have a high xylose content, the potential economic and ecologic benefits of converting xylose in these renewable materials to ethanol are significant.
In biomass conversion, microorganisms serve as biocatalysts to convert cellulosic materials into usable end products such as ethanol. Efficient biomass conversion in large-scale industrial applications requires a microorganism that can tolerate high sugar and ethanol concentrations, and which is able to ferment multiple sugars simultaneously.
The pentose D-xylose is significantly more difficult to ferment than D-glucose. Bacteria can ferment pentoses to ethanol and other co-products, and bacteria with improved ethanol production from pentose sugars have been genetically engineered. However, these bacteria are sensitive to low pH and high concentrations of ethanol, their use in fermentations is associated with co-product formation, and the level of ethanol produced remains too low to make the use of these bacteria in large-scale ethanol production economically feasible.
In general, industrial producers of ethanol strongly favor using yeast as biocatalysts, because yeast fermentations are relatively resistant to contamination, are relatively insensitive to low pH and ethanol, and are easier to handle in large-scale processing. Many different yeast species use xylose respiratively, but only a few species use xylose fermentatively. Fermentation of xylose to ethanol by wild type xylose-fermenting yeast species occurs slowly and results in low yields, relative to fermentation rates and ethanol yields that are obtained with conventional yeasts in glucose fermentations. In order to improve the cost effectiveness of xylose fermentation, it is necessary to increase the rate of fermentation and the ethanol yields obtained.
The most commonly used yeast in industrial applications is Saccharomyces cerevisiae. Although S. cerevisiae is unable to grow on or ferment xylose, homogenates of S. cerevisiae can readily ferment D-ribulose-5-phosphate to ethanol, and can convert D-xylulose-5-phosphate to a lesser extent. S. cerevisiae metabolically engineered to overproduce D-xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and D-xylulokinase (XYL3 or XKS1) or some forms of xylose isomerase (xylA) along with XYL3 or XKS1 can metabolize xylose to ethanol.
Pichia stipitis is a yeast species that in its native state is able to ferment xylose to produce ethanol. In P. stipitis, fermentative and respirative metabolism co-exist to support cell growth and the conversion of sugar to ethanol.
There is a need in the art for yeast strains having improved ability to convert sugars to ethanol.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides a nucleic acid construct comprising a coding sequence operably linked to a promoter not natively associated with the coding sequence. The coding sequence encodes a polypeptide having at least 95% amino acid identity to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
Other aspects of the invention provide yeast strains comprising the nucleic acid construct, and methods of producing ethanol or xylitol by growing a yeast strain of the invention in xylose-containing medium.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 shows the alignment of PsSUT2, PsSUT3, and PsSUT4.
FIG. 2 shows growth, measured as OD600, of S. cerevisiae expressing XUT1 or SUT4 on xylose as a function of time.
FIG. 3 shows growth, measured as OD600, of S. cerevisiae expressing XUT1 or SUT4 on glucose as a function of time.
FIG. 4 shows uptake of 14C-glucose or 14C-xylose, measured as DPM, by S. cerevisiae expressing XUT1 or SUT4 as a function of time.
FIG. 5 shows consumption of glucose and xylose by Pichia stipitis overexpressing SUT 4 as a function of time.
FIG. 6 shows glucose consumption and ethanol production by Pichia stipitis overexpressing SUT4 as a function of time.



DETAILED DESCRIPTION OF THE INVENTION
One approach to increasing the efficiency of yeast-catalyzed conversion of sugars to ethanol or to other products such as xylitol is to enhance uptake of sugars by the yeast. Cellulosic and hemicellulosic materials used in fermentation reactions are generally subjected to pretreatments and enzymatic processes that produce a mixture of several sugars, including glucose, xylose, mannose, galactose, and arabinose.
Saccharomyces cerevisiae and other hexose-fermenting yeasts have sugar transporters that facilitate uptake of hexoses. Some hexose sugar transporters also promote uptake of pentoses such as xylose, but at a much lower rate and with much lower affinity. Therefore, xylose uptake and utilization is impaired until glucose is consumed. When the substrate includes large initial concentrations of glucose, ethanol production from fermentation of glucose may result in inhibitory concentrations of ethanol before xylose uptake begins.
In order to promote xylose utilization by yeast in fermentation of mixed hemicellulosic sugars, sugar transport proteins having high affinity or specificity for xylose were identified and used to develop yeast engineered to have improved xylose uptake, as described below in the Examples.
The genome of the xylose-fermenting yeast Pichia stipitis was examined for putative sugar transporters based on similarity to transporters that facilitate uptake of hexoses, lactose, maltose, phosphate, urea, and other compounds. Eight putative xylose transporters (XUT1-7 and SUT4) were chosen based on their implied structures and sequence similarities to other reported sugar transporters. Genome array expression studies were used to evaluate whether the sugar transporters showed induced expression in cells grown on xylose. Genome array expression results were confirmed using quantitative PCR of transcripts from cells grown on xylose.
Sequences encoding polypeptides corresponding to those encoded by two genes (XUT1 and SUT4) that showed increased expression in cells grown on xylose-containing media under oxygen limited conditions were expressed in a host strain Saccharomyces cerevisiae JYB3011, which lacks all hexose-transport genes and expresses P. stipitis XYL1, XYL2, and XYL3, which encode xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK), respectively. Expression of either XUT1 or SUT4 allowed growth of the S. cerevisiae strain on xylose. Both XUT1 and SUT4 allowed increased xylose uptake. Cells carrying these sequences were tested for their relative affinities in taking up 14C-labeled glucose or 14C-labeled xylose. Xut1 was shown to have a higher affinity for xylose, and Sut4 a higher affinity for glucose. A transformant carrying a partial sequence of the XUT1 gene, which encodes the C-terminal portion of the protein, beginning with amino acid residue 154 of SEQ ID NO:2, was shown to bind xylose and glucose but did not accumulate these sugars, thereby demonstrating that at least some portion of the amino terminus of the protein is needed for sugar transport.
Thus, expression of any of XUT1, XUT1 partial, and SUT4 in Saccharomyces cerevisiae allows growth on xylose, and in the case of XUT1 and SUT4, uptake of xylose. It is envisioned that expression of any of XUT1, XUT1 partial, and SUT4 or sequences similar to XUT1, XUT1 partial, and SUT4 in other yeast species will also allow growth on xylose and/or uptake of xylose.
The sequence of the deduced amino acid sequence of the putative proteins encoded by the Pichia stipitis XUT1 and SUT4 genes are provided in SEQ ID NO:2 and SEQ ID NO:4, respectively. The sequences of the XUT1 and SUT4 coding sequences prepared from Pichia stipitis are shown in SEQ ID NO:1 and SEQ ID NO:3, respectively.
The P. stipitis glucose/xylose facilitators PsSut1-4 have a high degree of sequence identity, with PsSut1 having 78% amino acid identity with PsSut2, 3, and 4. PsSut2 differs from PsSut3 by one amino acid (L279V). PsSut4 differs from PsSut2 by just one amino acid (A544T) and from PsSUT3 by two amino acids (L279V and A544T). While all four facilitators have similar affinities for glucose, their affinities for xylose were shown to differ significantly (See Table 3). Notably, PsSut4 has a higher specificity ratio (V.sub.max/K.sub.m) than the other Sut facilitators for glucose and xylose, and a higher affinity for xylose than any of the other facilitators.
It is envisioned that changes to the polypeptides could be made without affecting their activities, and such variations are within the scope of the invention. For example, polypeptides have insertions, deletions or substitutions of amino acids relative to SEQ ID NO:2 and SEQ ID NO:4 are within the scope of the invention, provided that the polypeptide has at least 80% amino acid identity to SEQ ID NO:2 and SEQ ID NO:4. Suitably, a polypeptide according to the invention has at least 85%, 90%, or 95% amino acid identity to SEQ ID NO:2 or SEQ ID NO:4. It is further envisioned that polypeptides according to the invention have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity to SEQ ID NO:4, and have a threonine residue or a serine residue at a position corresponding to amino acid 544 of SEQ ID NO:4. Preferably, polypeptides according to the invention retain their activity, i.e., retain the ability to enhance uptake of glucose and/or xylose. Such activity may be assayed by any suitable means, including, for example, those described in the examples.
It is well understood among those of ordinary skill in the art that certain changes in nucleic acid sequence make little or no difference to the overall function of the protein or peptide encoded by the sequence. Due to the degeneracy of the genetic code, particularly in the third position of codons, changes in the nucleic acid sequence may not result in a different amino acid being specified by that codon. Changes that result in an amino acid substitution may have little or no effect on the three dimensional structure or function of the encoded protein or peptide. Conservative substitutions are even less likely to result in a lost of function. In addition, changes that result in insertions or deletions of amino acids may also be acceptable.
Alignment of the native sequence encoding PsSut4 with those encoding PsSut2 and PsSut3 shows that PsSut4 differs from the coding sequences for PsSut2 and PsSut3 at more than 30 bases (FIG. 1), with only two differences resulting in a change in the amino acid specified. Suitably, polynucleotides according to the invention include one or more of the divergent sequences of PsSUT4 shown in FIG. 1, in any combination. Suitably, the polynucleotides include anywhere from one to all of the divergent sequences of PsSUT4 shown in FIG. 1.
It is expected that expression of more than one of the proteins encoded by the polynucleotides of the invention, as exemplified by SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, could be obtained in a transgenic yeast cell. Expression of two or more polynucleotides of the invention may afford additional advantages in terms of fermentation rate or sugar uptake. For example, one could create a yeast strain that expresses a polynucleotide encoding a polypeptide having at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity to SEQ ID NO:2 together with a polynucleotide encoding a polypeptide having at least 80%, at least 85%, at least 90%, or at least 95% amino acid identity to SEQ ID NO:4.
Additionally, the polynucleotides of the invention may further include a sequence encoding a selectable marker that would allow selection of yeast expressing the polynucleotide.
In the examples, expression of SUT4 under the control of the promoter from P. stipitis fatty acid synthase 2 (FAS2 promoter), which is induced by xylose and under oxygen limiting conditions, was obtained in S. cerevisiae yJML123 and in P. stipitis UC7. The yJML123+SUT4 was able to use both glucose and xylose at a faster rate than the control strain. Neither strain was able to use all of the xylose. The yJML123+SUT4 strain, which has reduced expression of xylitol reductase, produced a higher yield of xylitol (18.45 g/L) than the control strain (15.51 g/L). Further, the yJML123+SUT4 strain produced a higher yield of xylitol faster than the control.
The P. stipitis UC7+SUT4 strain was grown under oxygen limiting conditions (50 mL of culture with a starting OD600 of 10 in a 125 mL flask aerated at speed of 100 rpm at 30° C.). The UC7+SUT4 strain was found to use xylose at a faster than the UC7 control; neither strain used all of the xylose. The xylitol yield of UC7+SUT4 strain (14.98 g/L) was similar to that of the UC7 control (15.18 g/L). The UC7+SUT4 strain had a higher ethanol specific productivity.
In another example, UC7+SUT4 strain was grown in glucose (7% w/w) and xylose (3% w/w) in a three liter bioreactor. Those results indicated that the UC7+SUT4 grew at a faster rate than did the UC7 control. The UC7+SUT4 strain had a higher biomass yield than the control. Additionally, the ethanol yield was higher for the UC7+SUT4 strain. The UC7+SUT4 strain consumed both glucose and xylose at a higher rate and consumed all of the sugars, whereas the control consumed only 88% of the glucose and did not appear to consume xylose. Finally, the specific rate of ethanol production on a dry cell weight basis was increased by more than three fold.
In the examples, strains were developed by causing nucleic acid of the invention to be introduced into a chromosome of the host yeast strain. This can be accomplished by any suitable means. However, it is envisioned that one could obtain increased expression of the nucleic acid constructs of the invention using an extrachromosomal genetic element, by integrating additional copies, by increasing promoter strength, or by increasing the efficiency of translation through codon optimization, all methods known to one of skill in the art.
It is envisioned that in addition to the particular strains that were used, any strain of S. cerevisiae or P. stipitis could be used in the practice of the invention. For example, in addition to P. stipitis UC7, one could advantageously obtain expression of the polynucleotides of the invention in P. stipitis strains having reduced respiration capacity, such as petite mutants or P. stipitis Shi21 (NRRL Y-21971). Similarly, one could advantageously obtain expression of the polynucleotides of the invention in a PHO-13 mutant of S. cerevisiae expressing P. stipitis XYL1, XYL2, and XYL3. Such a strain was developed, as described in the examples, for use in developing yeast strains according to the invention. Yeasts strains may suitably be modified to include exogenous sequences expressing Xut1 and Sut4 on the same or different nucleic acid. The yeast strains may be modified to include additional advantageous features, as would be appreciated by one skilled in the art.
In addition to S. cerevisiae and P. stipitis, it is envisioned that other yeast species could be used to obtain yeast strains according to the invention for use in the methods of the invention. Other suitable yeast species include, without limitation, Candida boidinii, Enteroramus dimorphus, Candida jeffriesii, Debaryomyces hansenii, Candida Guillermondii, Candida shehatae, Brettanomyces naardensis, Candida guillermondii, Candida lyxosophilia, Candida intermedia, Candida tenuis, Hansenula polymorpha, Kluyveromyces marxianus, Kluyveromyces lactis, Kluyveromyces fragilis, Kluyveromyces thermotolerans, Pachysolen tannophilus, Pichia segobiensis, Spathaspora passalidarum and Pass 1 isolates.
In another aspect, the present invention provides a method of fermenting xylose in a xylose-containing material to produce ethanol using the yeast of the invention as a biocatalyst. Another aspect of the present invention provides a method of fermenting xylose in a xylose-containing material to produce xylitol using the yeast of the invention as a biocatalyst. In this embodiment, the yeast preferably has reduced xylitol dehydrogenase activity such that xylitol is accumulated. Preferably, the yeast is recovered after the xylose in the medium is fermented to ethanol and used in subsequent fermentations.
The constructs of the invention comprise a coding sequence operably connected to a promoter. Preferably, the promoter is a constitutive promoter functional in yeast, or an inducible promoter that is induced under conditions favorable to uptake of sugars or to permit fermentation. Inducible promoters may include, for example, a promoter that is enhanced in response to particular sugars, or in response to oxygen limited conditions, such as the FAS2 promoter used in the examples. Examples of other suitable promoters include promoters associated with genes encoding P. stipitis proteins which are induced in response to xylose under oxygen limiting conditions, including, but not limited to, myo-inositol 2-dehydrogenase (MOR1), aminotransferase (YOD1), guanine deaminase (GAH1). These proteins correspond to protein identification numbers 64256, 35479, and 36448 on the Joint Genome Institute Pichia stipitis web site: genome.jgi-psf.org/Picst3/Picst3.home.html.
Oxygen limiting conditions include conditions that favor fermentation. Such conditions, which are neither strictly anaerobic nor fully aerobic, can be achieved, for example, by growing liquid cultures with reduced aeration, i.e., by reducing shaking, by increasing the ratio of the culture volume to flask volume, by inoculating a culture medium with a number of yeast effective to provide a sufficiently concentrated initial culture to reduce oxygen availability, e.g., to provide an initial cell density of 1.0 g/l dry wt of cells.
By “xylose-containing material,” it is meant any medium comprising xylose, whether liquid or solid. Suitable xylose-containing materials include, but are not limited to, hydrolysates of polysaccharide or lignocellulosic biomass such as corn hulls, wood, paper, agricultural by-products, and the like.
By a “hydrolysate” as used herein, it is meant a polysaccharide that has been depolymerized through the addition of water to form mono and oligosaccharides. Hydrolysates may be produced by enzymatic or acid hydrolysis of the polysaccharide-containing material, by a combination of enzymatic and acid hydrolysis, or by an other suitable means.
Preferably, the yeast strain is able to grow under conditions similar to those found in industrial sources of xylose. The method of the present invention would be most economical when the xylose-containing material can be inoculated with the mutant yeast without excessive manipulation. By way of example, the pulping industry generates large amounts of cellulosic waste. Saccharification of the cellulose by acid hydrolysis yields hexoses and pentoses that can be used in fermentation reactions. However, the hydrolysate or sulfite liquor contains high concentrations of sulfite and phenolic inhibitors naturally present in the wood which inhibit or prevent the growth of most organisms. Serially subculturing yeast selects for strains that are better able to grow in the presence of sulfite or phenolic inhibitors.
It is expected that yeast strains of the present invention may be further manipulated to achieve other desirable characteristics, or even higher specific ethanol yields. For example, selection of mutant yeast strains by serially cultivating the mutant yeast strains of the present invention on medium containing hydrolysate may result in improved yeast with enhanced fermentation rates.
The following non limiting examples are intended to be purely illustrative.
Examples
Characterization of Putative Xylose Transporters of Pichia stipitis 
Yeast strains, plasmids, and growth conditions. The yeast strains and the plasmids used in this study are listed in Table 1. S. cerevisiae EBY.VW4000 strain (MATa Δhxt1-17 Δgal2 Δstl1 Δagt1 Δmph2 Δmph3 leu2-3,112 ura3-52 trp1-289 his3-Δ1 MAL2-8c SUC2) (9) was kindly provided by E. Boles at Heinrich-Heine-Universität and was used for transformation of all the constructed plasmids. Three xylose transporter candidates, XUT1, XUT1 partial (corresponding to the sequence encoding the C-terminal region of the protein beginning with amino acid residue 154 of SEQ ID NO:2), and SUT4, were amplified and fused with the TDH3 promoter by PCR. The coding sequence of SUT4 includes a CUG codon, which in P. stipitis, specifies the serine residue of amino residue 410 of SEQ ID NO:4. This codon, which in most organisms specifies a leucine, was modified for expression in S. cerevisiae using standard methods so as to specify a serine residue. Each candidate had its own terminator. These fragments were then inserted into pRS316 after sequence confirmation (Sikorski & Hieter (1989) Genetics 122, 19-27). Genetic manipulation and cloning of DNA were performed using methods known in the art. Escherichia coli DH5αF′ was used as a host for plasmid preparation.
Yeast transformation was performed as previously described (Gietz & Woods (2002) Methods Enzymol 350, 87-96). Yeast transformants were selected by TRP1 and URA3 selectable markers and cultivated on yeast synthetic complete dropout (TRP-URA-) medium with carbon source (Kaiser et al. & Cold Spring Harbor Laboratory. (1994) Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)). Yeast cells were cultured in a 125-ml flask with 50 ml medium at 30° C. and 200 rpm.
Quantitative PCR (qPCR)
Total RNA was prepared from each transformant and 5 μg of total RNA was used for cDNA construction with random oligonucleotides and the Reverse Transcription System Kit (Promega, Madison, Wis.). Reverse Transcription-PCR primers were designed using Primer Express software (Applied Biosystems). RT-PCR was performed with SYBR green PCR master mix (Applied Biosystems) and an ABI PRISM 7000 sequence detection system (Applied Biosystems). PCR conditions were as recommended by the manufacturer except that one-half of the reaction volume was used. Actin was used to normalize the relative copy numbers of each gene. All reactions were performed in triplicate.







TABLE 1







List of yeast strains and plasmids











Source or


Strain or plasmid
Description
reference





Strains




S. cerevisiae
MATa Δhxt1-17 Δgal2 Δstl1 Δagt1 
Wieczorke


EBY.VW4000
Δmph2 Δmph3 leu2-3, 112 ura3-52 




trp1-289 his3-Δ1 MAL2-8c SUC2



S. cerevisiae
S. cerevisiae EBY.VW4000
This study


JYB3005
(pRS314 and pRS316)



S. cerevisiae
S. cerevisiae EBY.VW4000
This study


JYB3011
(pRS314-X123 and pRS316)



S. cerevisiae
S. cerevisiae EBY.VW4000
This study


JYB3110
(pRS314-X123 and 




pRS316-XUT1partial)



S. cerevisiae
S. cerevisiae EBY.VW4000
This study


JYB3210
(pRS314-X123 and pRS316-XUT1)



S. cerevisiae
S. cerevisiae EBY.VW4000
This study


JYB3310
(pRS314-X123 and pRS316-SUT4)



Plasmids




pRS314
TRP1 CEN/ARS
Sikorski


pRS316
URA3 CEN/ARS
Sikorski


pRS314-X123
TRP1 CEN/ARS 
Ni



TDH3P-PsXYL1-TDH3T 




TDH3P-PsXYL2-TDH3T 




TDH3P-PsXYL3-TDH3T



pRS316-
URA3 CEN/ARS TDH3P-PsXUT1
This study


XUT1partial




pRS316-XUT1
URA3 CEN/ARS TDH3P-PsXUT1
This study


pRS316-SUT4
URA3 CEN/ARS TDH3P-PsSUT4
This study









Sugar-Transport Assay.
Initial D-[14C]xylose and D-[14C]glucose (Amersham) uptake rates were measured at 30° C., pH 5.0 as previously described (Spencer-Martins, I. a. c. U., N. (1985) Biochim. Biophys. Acta 812, 168-172). Cells were harvested in the mid-exponential phase (A600 nm 0.8-1.0; ≈0.1-0.125 g/l dry wt) by centrifugation, washed twice with ice-cold water and resuspended to a cell concentration of 5-10 mg (dry weight)/ml. Kinetic parameters were determined using Lineweaver-Burk plots. All measurements were conducted at least three times, and the data reported are the average values.
Transcriptional Analysis of Eight Xylose Transporter Candidates.
Eight putative xylose transporters (XUT1-7 and SUT4) were identified based on genomic analysis of the genome sequence of Pichia stipitis (Jeffries et al. (2007) Nat Biotechnol 25, 319-26) and selected for further analysis. Of these eight putative transporters, XUT1, and SUT4 showed increased expression in Pichia stipitis grown in xylose-containing media. These two genes were introduced into the host strain S. cerevisiae JYB3011 to evaluate the ability of the putative xylose transporters to promote xylose uptake. This host strain was originated from S. cerevisiae EBY.VW4000 (Wieczorke et al. (1999) FEBS Lett 464, 123-8) and engineered to express PsXYL1, PsXYL2, and PsXYL3 by introducing pRS314-X123 (Ni et al. (2007) Appl Environ Microbiol 73, 2061-6) (Table 1).
Kinetic Characteristics of Sugar Uptake.
XUT1 partial, XUT1 and SUT4 were expressed in the strains S. cerevisiae JYB3110, JYB3210, and JYB3310, respectively (Table 1). The expression of each transporter in its respective strain was confirmed by qPCR (data not shown). These three strains could grow in xylose-containing media (FIG. 2) and in glucose-containing media (FIG. 3). Xylose and glucose transport capacity of each candidate was tested using a high concentration (50 mM) of labeled sugar (Table 2). The strains containing Xut1 or Sut4 showed increased xylose uptake activities, with the strain expressing Xut1 having a higher uptake rate than that of the strain expressing Sut4 (FIG. 4). Both JYB 3210 and JYB3310 showed glucose-transporting activity, with Sut4 conferring a higher glucose uptake velocity than Xut1 (FIG. 4).
Kinetic analysis demonstrated that the affinity of Xut1 for xylose is 3-fold higher than its affinity for glucose (FIG. 4 and Table 3). This is the first reported transporter having a higher affinity for xylose. Vmax was also higher with xylose than glucose. Although Sut4 exhibits xylose uptake activity, its affinity for glucose is 12 times higher than its affinity for xylose (Table 3). Interestingly, Sut4 has higher Vmax with xylose than glucose. Notably, Sut4 has a higher specificity ratio (Vmax/Km) for both xylose and glucose than any of the other four glucose/xylose facilitators found in Pichia stipitis (Table 3).
Table 3 compares Km values of the newly discovered xylose transporters to previously characterized xylose transporters. All four of the PsSut glucose/xylose facilitators show similar affinities for glucose, but their affinities for xylose differ significantly. Notably, PsSut4 shows a higher specificity ratio (Vmax/Km) for glucose and xylose than the specificity ratios of PsSut1-3. Interestingly, PsSut1 has about 78% amino acid identity with PsSut2, 3 and 4, whereas PsSut2 and PsSut4 differ from PsSut3 by one or two amino acids each (Sut2: L279V; Sut4: L279V, A544T). Despite the high degree of sequence identity, PsSut1-4 have distinct properties. PsSut1 and PsSut3 can mediate fructose transport, whereas PsSut2 cannot, and only PsSut3 can mediate galactose transport (Weierstall et al. (1999) Molecular Microbiol. 31:871-883). Notably, among PsSut1-4, PsSut4 has the highest affinity and the second highest Vmax for xylose. The affinity of PsSut4 for xylose is about three times higher than that of PsSut2, and its Vmax for xylose uptake is about 4.4 times higher than the Vmax of Sut2.
Energy requirements for sugar transport were evaluated by including the metabolic uncoupler sodium azide. Xut1 mediated uptake of sugar was reduced by 96% in the presence of sodium azide, whereas Sut4 mediated sugar uptake was reduced by about 50% (Table 4). Consequently, Xut1 is believed to be a high affinity xylose-proton symporter, and SUT4 appears to employ facilitated diffusion.







TABLE 2







Initial uptake rates of labeled sugar (50 mM) of 


each putative xylose transporter.











Strain
Substrate
V‡















JYB3011 (Control) 
Glucose
5




Xylose
0



JYB3110 (XUT1
Glucose
7



partial)






Xylose
0



JYB3210 (XUT1)
Glucose
31.8




Xylose
57.8



JYB3310 (SUT4)
Glucose
52.6




Xylose
23.1







‡Velocity of uptake (milimoles per minutes per milligram [dry weight])













TABLE 3







Kinetic parameters of yeast glucose/xylose transporters












Km
Vmax
Specificity




(mM)
(nmol min−1 mg dw−1)
Vmax/Km















Transporters
Glucose
Xylose
Glucose
Xylose
Glucose
Xylose
Reference





















PsSut1
1.5 ±
0.1
145.0 ±
1.0
45 ±
1.0
132 ±
1.0
30.0
0.9
(4)


PsSut3
0.8 ±
0.1
103 ±
3.0
3.7 ±
0.1
87 ±
2.0
4.6
0.8
(4)


PsSut2
1.1 ±
0.1
49.0 ±
1.0
3.3 ±
0.1
28 ±
4.0
3.3
0.6
(4)


PsSut4
1.3 ±
0.1
16.6 ±
0.3
105 ±
4.2
122 ±
2.4
80.8
7.4
This study
















CiGxf1
2.0 ±
0.6
48.7 ±
6.5
≈163†
≈25†
81.5
0.5
(1)


















ScHxt1
a107 ±
49
b880 ±
8
a50.9 ±
3.7
b750 ±
94
0.5
0.8
a(2) b(3)


ScHxt2
a2.9 ±
0.3
b260 ±
130
a15.6 ±
0.9
b340 ±
10
5.4
1.3
a(2) b(3)


ScHxt4
a6.2 ±
0.5
b170 ±
120
a12.0 ±
0.9
b190 ±
23
1.9
1.1
a(2) b(3)


ScHxt7
a1.3 ±
0.3
b130 ±
9
a11.7 ±
0.3
b110 ±
7
90.0
0.8
a(2) b(3)


CiGxs1
0.012 ±
0.004
0.4 ±
0.1
4.3 ±
.33
6.5 ±
1.5
358.3
16.2
(1)


PsXut1
0.91 ±
0.01
0.46 ±
0.02
80 ±
1.0
116 ±
5.8
87.91
252.2
This study





†Calculated from FIG. 2 of (1)


1. Leandro, M. J., P. GonÁalves, and I. Spencer-Martins. 2006. Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose-H+ symporter. The Biochemical journal 395:543-549.


2. Maier, A., B. Volker, E. Boles, and G. F. Fuhrmann. 2002. Characterisation of glucose transport in Saccharomyces cerevisiae with plasma membrane vesicles (countertransport) and intact cells (initial uptake) with single Hxtl, Hxt2, Hxt3, Hxt4, Hxt6, Hxt7 or Gal2 transporters. Fems Yeast Research 2:539-550.


3. Saloheimo, A., J. Rauta, O. V. Stasyk, A. A. Sibirny, M. Penttila, and L. Ruohonen. 2007. Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases. Applied Microbiology And Biotechnology 74:1041-1052.


4. Weierstall, T., C. P. Hollenberg, and E. Boles. 1999. Cloning and characterization of three genes (SUT1-3) encoding glucose transporters of the yeast Pichia stipitis. Molecular Microbiology 31:871-883.













TABLE 4







Effect of sodium azide on glucose and xylose uptake‡.












Glucose
Xylose















NaN3 

NaN3 




Control
(50 mM)
Control
(50 mM)

















JYB3110 (SUT4)
41
0.32
90
3.19



JYB3310 (XUT1)
40
25
13.4
9.2







‡Velocity of uptake (nmol min−1 mg−1 [dry weight])






Effect of Expression of Sut4 in P. stipitis 

Construction of Plasmids and Transformation of Yeast Strains.
A plasmid was constructed to contain the P. stipitis coding sequence for the SUT4 putative transporter under the control of the P. stipitis fatty acid synthase subunit alpha (Fas2) promoter (FAS2p_SUT4_SUT4t_LoxP_URA3_LoxP plasmid). Roughly 100 ug of the FAS2p_SUT4_SUT4t_LoxP_Ura3_LoxP plasmid was linearized using Hind III and ApaI, ethanol precipitated and resuspended in water to create a fragment that would integrate directly into the Pichia genome. The digested construct was then used in a LiAc protocol and transformed into either the yJML123 (Gietz & Woods (2002) Methods Enzymol 350, 87-96)) or UC7 Pichia stipitis strains. The yJML123 strain has a deletion in the XYL2 gene, which encodes the enzyme that catalyzes the conversion of xylitol to xylulose, allowing us to create Pichia strains that will begin to accumulate xylitol.
Transformants were selected for via growth on ScD-Ura plates, which contain 0.62 g/L CSM-Leu-Trp-Ura (Bio 101 Systems) and dextrose (2%). Colonies were then picked and grown overnight in ScD-Ura liquid media. Genomic DNA was then extracted and evaluated by PCR to confirm integration of the construct. As a control for these strains, a plasmid containing only the LoxP_Ura3_LoxP cassette was also integrated into the yJML123 and UC7 strains (yJML123 and UC7 controls).
Fermentation of yJML123+SUT4
Starter cultures were established by inoculating a single colony of yJML123+SUT4 strain or yJML123 control from recently streaked plate into 25 mL fermentation media and grown overnight. The following morning, triplicate flask cultures of each transformant were started at an OD600 of 0.5 (≈0.06 g/l dry wt of cells). The transformants were confirmed using PCR genotyping. The fermentation was run under aerobic conditions using an agitation speed of 200 rpm at 30° C. in 25 mL of media in a 125 mL flask in fermentation medium (Yeast Nitrogen Base (1.7 g/L), urea (2.4 g/L), peptone (3.6 g/L), with xylose (40 g/L) and glucose (17 g/L) as carbon sources. Samples were collected for analysis roughly every 8 hours for the first 48 hours and then once every 24 hours.
The yJML123+SUT4 strain was able to use both glucose and xylose at a faster rate than the control strain. Neither strain was able to use all of the xylose. The yJML123+SUT4 strain produced a higher yield of xylitol (18.45 g/L) than the control strain (15.51 g/L). Further, the yJML123+SUT4 strain produced a higher yield of xylitol faster than the control.
Fermentation of UC7+SUT4.
Starter cultures were established by inoculating a single colony of SUT4+UC7 strain or the UC7 control from recently streaked plates into 50 mL of fermentation media and grown overnight. The following morning, triplicate flasks of each transformant were started at an OD600 of 0.5 (≈0.06 g/l dry wt of cells). The transformants were confirmed using PCR genotyping. The fermentation was run in fermentation medium (Yeast Nitrogen Base (1.7 g/L), urea (2.4 g/L), peptone (3.6 g/L), and xylose (40 g/L), under low oxygen conditions using an agitation speed of 100 rpm at 30° C. in 50 mL of media in a 125 mL flask. The starting OD600 was 10 (≈1.25 g/l dry wt of cells). Samples were collected for analysis roughly every 12 hours for the first 60 hours and then once every 24 hours.
Fermentation of UC7+SUT4.
The UC7+SUT4 strain was found to use xylose at a faster rate than the UC7 control; neither strain used all of the xylose. The xylitol yield of UC7+SUT4 strain was similar to that of the UC7 control. The UC7+SUT4 strain had a higher ethanol specific productivity than that of the UC7 control.
Evaluation of Expression of SUT4 in P. stipitis in Bioreactor
Strain Descriptions.
The UC7+SUT4 strain described above (“SUT4”) and the UC7+URA3 cassette strain (“control”) described above were evaluated in a three liter bioreactor scale-up as described below.
3 L Bioreactor Scale-up of the SUT4 Transporter Overexpression Strain.
Low oxygen bioreactor trials were used to compare two strains in a larger scale and under more controlled conditions than shake flask trials.
Bioreactor Cultivations.
A defined minimal medium containing trace metal elements and vitamins was used in all bioreactor cultivations. This medium was modified based on that described by Verduyn et al. (Verduyn et al. (1992) Yeast 8:501-517) to include urea as a nitrogen source. It had the following composition: 2.4 g urea L−1; 3 g KH2PO4 L−1, 0.5 g MgSO4 7 H2O L−1; 1 ml trace element solution L−1; 1 ml vitamin solution L−1; and 0.05 ml antifoam 289 (Sigma A-8436) L−1 (Jeffries & Jin (2000) Adv Appl Microbiol 47, 221-68). For these cultivations, a starting concentration of 70 g L−1 glucose and 30 g L−1 xylose was used.
Cultivations were performed in 3 L New Brunswick Scientific BioFlo 110 Bioreactors with working volumes of 2 liters. All cultivations were performed at 30° C. Agitation was set at 500 rpm and the pH was controlled at pH 5.0 by the addition of 5 N KOH and 5 N H2SO4. Aeration was controlled at a rate of 0.5 vvm which corresponded to a rate of 1 L min−1. Input gas was mixed using a gas proportioner to include 90% pure nitrogen and 10% air, for a final oxygen concentration of approximately 2%. Bioreactors were inoculated to an OD of approximately 1 and their progress followed for 74 hours.
During cultivation, the SUT4 strain appeared to grow faster than the control. Calculated biomass rates and yields demonstrated that SUT4 had a slightly improved growth rate ( ̃1.2 fold) (0.062 vs. 0.0514 h−1) and biomass yield (0.151 g dry cell weight/g glucose vs. 0.145 g dry cell weight/g glucose, or 0.184 Cmmol vs. 0.177 Cmmol). As was observed previously in the shake flask experiments described above, the overall difference in ethanol yield from glucose was not large. The SUT4 strain had a slightly higher ( ̃1.11 fold) ethanol yield on glucose (0.372 g ethanol/g glucose or 0.485 Cmmol) than the control strain (0.335 g ethanol/g glucose or 0.437 Cmmol/Cmmol glucose).
The major difference between these two strains is in their kinetic rates of glucose and xylose consumption. The SUT4 strain consumes glucose at a much higher rate than the control strain (FIG. 5). In fact, the SUT4 strain consumed all of the glucose and xylose in the fermentation, while the URA3 control strain consumed approximately 88% of the glucose and did not consume enough xylose to significantly change the xylose concentration during the length of the fermentation.
The specific rate of glucose consumption for the SUT4 strain (0.284 g glucose gDCW−1 h−1 or 9.448 Cmmol gDCW−1 h−1) was 3.04 fold higher than that of the URA3 control strain (0.093 g Glucose gDCW−1 h−1 or 3.106 Cmmol gDCW−1 h−1). The specific rates of xylose consumption could not be compared due to the fact that the URA3 control strain had not significantly begun to utilize the xylose. Further bioreactor trials will be necessary to compare these rates.
The SUT4 and control strains also exhibited different rates of ethanol production. FIG. 6. shows a plot of ethanol production and glucose consumption. It should be noted that a small amount of ethanol produced by the SUT4 overexpression strain was due to the xylose consumed; however the rates and yields were calculated using the glucose only phase of fermentation. The specific rate of ethanol production for the SUT4 overexpression strain (0.106 g ethanol gDCW−1 or 4.580 Cmmol gDCW−1 h−1) was 3.38 fold higher than that of the control strain (0.031 g ETOH gDCW−1 h−1 or 1.356 Cmmol gDCW−1 h−1).
Creation of the Δpho13 Background Strains for Transporter Testing
In order to test the effect of putative transporters on xylose utilization, a robust Saccharomyces cerevisiae background that is capable of utilizing xylose in some capacity needed to be built. Previous research has shown that either the overexpression of TAL1 or the deletion of PHO13 promotes growth on xylose when the Pichia stipitis xylulokinase gene is expressed at a high level (10). The PHO13 deletion was chosen for use in these strains, as more detailed kinetic data during aerobic and low oxygen cultivations is available for a strain created in a similar genetic background (Van Vleet et al. (2008) Metab. Eng. Doi10.1016/j.ymben.2007.12.002).
The strains CEN.PK.111-27B and CEN.PK.102-3A were chosen as the genetic backgrounds in which to create these deletion strains (Entian K, Kötter P, (2007) 25 Yeast Genetic Strain and Plasmid Collections. In: Methods in Microbiology; Yeast Gene Analysis—Second Edition, Vol. Volume 36 (Ian Stansfield and Michael JR Stark ed), pp 629-666. Academic Press). The CEN.PK.111-27B carries the trp1 and leu2 mutations and the CEN.PK.102-3A strain carries the ura3 and leu2 mutations. The deletion of the PHO13 in these backgrounds will allow for the putative transporters to be tested in the presence of the xylose utilization genes expressed moderately and, separately, at a high level. These transporters will also be able to be tested at 2 different levels of expression. Moderate expression of the transporters will be possible using a centromeric LEU2 plasmid and high levels of expression will be possible using a multicopy LEU2 plasmid with a 2 μm origin of replication.
The PHO13 deletion cassette was amplified out of the genomic DNA of S. cerevisiae CMB.JHV.pho13a (Van Vleet et al. (2008) Metab. Eng. Doi10.1016/j.ymben.2007.12.002) using the high fidelity Pfusion polymerase (NEB, Finnzymes) and primers designed 400 bp upstream and downstream of the PHO13 open reading frame. This cassette contains the KANMX gene and has been shown previously to confer G418 resistance to PHO13 knockout strains in S. cerevisiae (Van Vleet et al. (2008) Metab. Eng. Doi10.1016/j.ymben.2007.12.002).
HpaI sites were added onto the ends of this amplified fragment for easy excision from a storage vector. The primers used in strain creation and confirmation are listed in Table 5. The amplified fragment was ligated into the pCR4 Blunt-TOPO vector and then transformed into TOP10 E. coli by a heat shock method. Transformants were selected for on kanamycin plates and digest checked to confirm the size of the insert. After verification, 100 μg of plasmid DNA was prepared using a Qiagen maxiprep and digested with HpaI to release the deletion cassette. The deletion cassette was then gel purified.
The purified deletion cassette was then transformed into the CEN.PK strains using a standard lithium acetate method (Gietz & Woods (2002) Methods Enzymol 350, 87-96)). Outgrowth was performed after the transformation for 3 hours in YPD liquid media, and then transformants were selected for on YPD plates containing 250 mg/ml G418. After 2 days of growth, growth of putative knockouts was confirmed by re-streaking on fresh G418 plates. These strains were then genotyped using primers that were designed 600 bp upstream and downstream of the PHO13 open reading frame. These primers amplify a product in both deletion and non deletion strains, however in deletion strains the product is approximately 650 bp larger than in the non deletion strain. Multiple transformants for each strain were identified as containing the deletion. Transformants containing the deletion originating from CEN.PK.111-27B were designated FPL.JHV.002 and those originating from CEN.PK.102-3A were designated FPL.JHV.003.
The xylose utilization genes (XYL1, XYL2, and XYL3) under the control of the strong constitutive TDH3 promoter were then introduced to the strains via plasmid. FPL.JHV.002 was transformed with the centromeric vector pRS314-X123 (10) and designated FPL.JHV.004. At the same time, FPL.JHV.003 was transformed with the multicopy vector pYES2-X123 (10) and designated FPL.JHV.005. Both of the vector bearing strains carry a leu2 marker to allow for testing of the putative transporters. The plasmids and yeast strains are described in Table 6.







TABLE 5







Primers Used









Primer Name
Function
Sequence





oFPL.JHV.021
PHO13F + HpaI
GATTACATGTTAACGATTGTTCGACGCAACTACCC




(SEQ ID NO: 8)


 


oFPL.JHV.022
PHO13R + HpaI
GATTACATGTTAACCTTCCCCAACAAGACCGAATTG




(SEQ ID NO: 9)


 


oFPL.JHV.023
PHO13 Confirmation F
TTAAAACAAGAATTTGGGGAG




(SEQ ID NO: 10)


 


oFPL.JHV.024
PHO13 Confirmation
AAAGGTCTAATTATTCAATTTATCGAC




(SEQ ID NO: 11)









Assessment of Putative Transporters by Whole Genome Expression Array Technology.
Cells of Pichia stipitis were cultivated in a bioreactor with either xylose or glucose as carbon sources along with mineral nutrients and urea for a nitrogen source using the medium of Verduyn et al. (16). Cells were grown either under fully aerobic or oxygen limited conditions. Cells were harvested, their mRNA was extracted and analyzed using a whole-genome expression array (NimbleGen). Transcripts corresponding to putative sugar transporters were identified and annotated. (Results not shown). Transcripts for seven genes (HXT2.4, HX′T4, SUC1.5, XUT1, SUT4, XUT7 and YBR2) were induced to a higher level in cells grown on xylose than in cells grown on glucose. All but SUT4 were induced to their highest levels on xylose under oxygen limiting conditions. Transcripts for seven genes (AUT1, HGT2, HUT1, MAL4, RGT2, STL1 and SUT1) were all induced to their highest levels when cells were cultivated on glucose. Transcript for one gene (SUT1) was induced to its highest level under oxygen limitation; the other six were induced to their highest levels under aerobic conditions. The specific induction of transcripts on xylose under aerobic or oxygen limited conditions was taken as an indicator of the effective function of this gene when cells were cultivated on xylose. Of these genes, SUT1, SUT2 and SUT3 were previously recognized and described as glucose/xylose facilitator transport proteins.
It is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
It also is understood that any numerical range recited herein includes all values from the lower value to the upper value. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

## Claims

1. A method of producing ethanol or xylitol from the fermentation of xylose comprising: culturing a yeast strain in contact with a xylose-containing material under suitable conditions for a period of time sufficient to allow fermentation of at least a portion of the xylose to ethanol or xylitol, wherein the yeast strain comprises a nucleic acid comprising a coding sequence operably linked to a promoter not natively associated with the coding sequence, the coding sequence encoding a glucose/xylose transporter polypeptide having at least 95% amino acid identity to SEQ ID NO:2, or at least 95% amino acid identity to SEQ ID NO:4 and comprising a threonine residue at the amino acid position corresponding to position 544 of SEQ ID NO:4.

2. The method of claim 1, wherein the strain exhibits increased uptake of xylose relative to a control yeast lacking the nucleic acid.

3. The method of claim 1, wherein at least a portion of the xylose is converted to ethanol.

4. The method of claim 1, wherein the xylose-containing material further comprises glucose.

5. The method of claim 1, wherein the promoter is a constitutive promoter or promoter inducible under oxygen limiting conditions.

6. The method of claim 1, wherein the polypeptide comprises SEQ ID NO:2.

7. The method of claim 1, wherein the coding sequence encodes a glucose/xylose transporter with 95% amino acid identity to SEQ ID NO:2 and a glucose/xylose transporter polypeptide having at least 95% amino acid identity to SEQ ID NO:4 and comprising a threonine or serine residue at the amino acid position corresponding to position 544 of SEQ ID NO:4.

8. The method of claim 1, wherein the polypeptide comprises SEQ ID NO:4.

9. The method of claim 1, wherein the yeast strain is a Pichia stipitis or Saccharomyces.

10. The method of claim 1, wherein at least a portion of the xylose is converted to xylitol.

11. The method of claim 10, wherein the strain exhibits increased xylitol production relative to a control yeast lacking the nucleic acid.

12. The method of claim 10, wherein the strain has reduced xylitol dehydrogenase activity such that xylitol accumulates.