Provisional Patent Application 60/082,334, filed April 20,1998, the disclosure of which is relied upon and incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention This invention relates to recombinant integration vectors. More specifically, this invention provides recombinant integration vectors containing sequences of a gene encoding an aldose reductase (AR), but not the entire AR gene. The recombinant vector can be used to specifically delete or disrupt the AR-encoding gene of a host cell. The recombinant vector also permits any heterologous sequence to be integrated into the host genomic AR sequence. Integration into the AR sequence of, for example a yeast strain, renders the recombinant strain less efficient at producing, or even unable to produce, xylitol from xylose. The recombinant vector can further be used to insert genes coding for xylose utilizing enzymes, which provides a recombinant strain which not only can utilize xylose but is simultaneously prevented from xylitol formation through the action of the AR.

Description of the Related Art Lignocellulose is the main component of forest product residues and agricultural waste. Lignocellulosic raw materials are mainly composed of cellulose, hemicellulose, and lignin. The cellulose fraction is made up of glucose polymers, whereas the hemicellulose fraction is made up of a mixture of glucose, galactose, mannose, xylose, and arabinose polymers. The lignin fraction is a polymer of phenolic compounds.

The cellulose and hemicellulose fractions can be hydrolyzed to monomeric sugars, which can be fermented to ethanol. Ethanol can serve as an environmentally friendly liquid fuel for transportation, since carbon dioxide released in the fermentation and combustion processes will be taken up by growing plants in forests and fields.

The price for lignocellulose-derived ethanol has been estimated by von Sivers et al. ("Cost analysis of ethanol production from willow using recombinant Escherichia cols', Biotechnol. Prog. 10: 555-560,1994). The calculations are based on the fermentation of all hexose sugars (glucose, galactose, and mannose) to ethanol. It was estimated that the fermentation of pentose sugars (xylose and arabinose) to ethanol will reduce the price of ethanol by approximately 25%.

Xylose is found in hardwood hemicellulose, whereas arabinose is a component in hemicellulose in certain agricultural crops, such as corn. In order to make the price more competitive, the price must be reduced.

The release of monomeric sugars from lignocellulosic raw materials also releases by-products, such as weak acids, furans, and phenolic compounds, which are inhibitory to the fermentation process. Numerous studies have shown that the commonly used Baker's yeast, Saccharomyces cerevisiae, is the only ethanol producing microorganism that is capable of efficiently fermenting non-detoxified lignocellulose hydrolysates (Olsson and Hahn-Hagerdal,"Fermentation of lignocellulosic hydrolysates for ethanol production", Enzyme Microbial Technol.

18: 312-331,1996). Particularly efficient fermenting strains of S. cerevisiae have been isolated from the fermentation plant at a pulp and paper mill (Lindén et al., "Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant", Appl.

Environ. Microbiol. 58: 1661-1669,1992).

S. cerevisiae ferments the hexose sugars glucose, galactose and mannose, but is unable to ferment the pentose sugars xylose and arabinose due to the lack of one or more enzymatic steps. S. cerevisiae can ferment xylulose, an isomerization product of xylose, to ethanol (Wang et al.,"Fermentation of a pentose by yeasts", Biochem. Biophys. Res. Commun. 94: 248-254,1980; Chiang et al.,"D-Xylulose fermentation to ethanol by Saccharomyces cerevisiae", Appl.

Environ. Microbiol. 42: 284-289,1981; Senac and Hahn-Hagerdal,"Intermediary metabolite concentrations in xylulose-and glucose-fermenting Saccharomyces cerevisiae cells", Appl. Environ. Microbiol. 56: 120-126,1990).

In eukaryotic cells, the initial metabolism of xylose is catalyzed by a xylose reductase (XR), which reduces xylose to xylitol, and a xylitol dehydrogenase (XDH), which oxidizes xylitol to xylulose. Xylulose is phosphorylated to xylulose 5- phosphate by a xylulose kinase (XK) and further metabolized through the pentose phosphate pathway and glycolysis to ethanol.

S. cerevisiae has been genetically engineered to metabolize and ferment xylose. The genes for XR and XDH from the xylose fermenting yeast Pichia stipitis have been expressed in S. cerevisiae (European Patent to C. Hollenberg, 1991; Hallborn et al., "Recombinant yeasts containing the DNA sequences coding for xylose reductase and xylitol dehydrogenase enzymes", W091/15588; Kotter and Ciriacy,"Xylose fermentation by Saccharomyces cerevisiae", Appl. Microbiol. Biotechnol. 38: 776- 783,1993). The transformants metabolize xylose but do not ferment the pentose sugar to ethanol.

When the gene for the enzyme transaldolase (TAL) is overexpressed in xylose-metabolizing transformants, the new recombinant strains grow better on xylose but still do not produce any ethanol from xylose (Walfridsson et al.,"Xylose- metabolizing Saccharomyces cerevisiae strains overexpressing the TKL 1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase", Appl. Environ. Microbiol. 61: 4184-4190,1995). In these strains, the major metabolic by-product, in addition to cell mass, is xylitol formed from xylose through the action of the enzyme XR. When the expression of XDH is ten times higher than the expression of XR, xylitol formation is reduced to zero (Walfridsson et al.,"Expression of different levels of enzymes from Pichia stipitis XYL1 and XYL2 genes in s and its effect on product formation during xylose utilization", Appl.

Microbiol Biotechnol. 48: 218-224,1997). However, xylose is still not fermented to ethanol.

The gene for xylulose kinase (XK) from S. cerevisiae has been cloned and overexpressed in XR-XDH-expressing transformants of S. cerevisiae (Deng and Ho,"Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene", Appl. Biochem. Biotechnol. 24/25: 193- 199,1990; Ho and Tsao,"Recombinant yeasts for effective fermentation of glucose and xylose", W095/13362,1995; Moniruzzaman et al.,"Fermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces strain", World J.

Microbiol. Biotechnol. 13: 341-346,1997). These strains have been shown to produce net quantities of ethanol in fermentations of mixtures of xylose and glucose. Using the well established ribosomal integration protocol, the genes have been chromosomally integrated to generate strains that can be used in complex media without selection pressure (Ho and Chen,"Stable recombinant yeasts for fermenting xylose to ethanol", W097/42307; Toon et al.,"Enhanced cofermentation of glucose and xylose by recombinant Saccharomyces yeast strains in batch and continuous operating modes", Appl. Biochem. Biotechnol.

63/65: 243-255,1997).

In prokaryotic cells, xylose is isomerized to xylulose by a xylose isomerase (XI). Xylulose is further metabolized in the same manner as in the eukaryotic cells.

Xi from the thermophiiic bacterium Thermus thermophilus was expressed in S. cerevisiae, and the recombinant strain fermented xylose to ethanol (Walfridsson et al.,"Ethanolic"Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene which expresses an active xylose (glucose) isomerase", Appl. Environ. Microbiol. 62: 4648-4651,1996). The low level of ethanol produced was assumed to be due to the fact that the temperature optimum of the enzyme is 85°C, whereas the optimum temperature for a yeast fermentation is 30°C.

Recently, the gene for XI from a mesophilic bacterium, Streptomyces diastaticus, has been cloned and transformed into S. cerevisiae. When xylose is fermented by an Xi expressing transformant of S. cerevisiae, a considerable amount of xylitol is formed in addition to ethanol. The xylitol is believed to be produced by an unspecified aldose reductase (AR) (Kuhn et al.,"Purification of an aldo-keto reductase from Saccharomyces cerevisiae", Appl. Environ. Microbiol.

61: 1580-1585,1995).

Although great strides have been made, there exists a need in the art for a method of efficiently fermenting lignocellulose hydrolysates to produce ethanol.

SUMMARY OF THE INVENTION In order fulfill the above-noted need, the present invention provides genetically engineered (recombinant) expression vectors, and recombinant cells capable of fermenting lignocellulose hydrolysates to ethanol. This invention aids in fulfilling a need in the art by providing integration vectors containing sequences of an aldose reductase (AR). The recombinant vectors can be used to specifically delete or disrupt an endogenous AR gene, and can also be used to incorporate heterologous polynucleotide sequences into host (recipient) cells. The recombinant vector constructs permit any gene, including those coding for xylose- utilizing enzymes, to be integrated in the AR gene sequence. In this way, recombinant cells are produced that show enhanced conversion of xylose to ethanol while simultaneously showing reduced xylitol formation through the action of the endogenous AR.

Accordingly, the invention provides methods of making recombinant cells and methods of efficiently producing ethanol from lignocellulose-containing compositions.

BRIEF DESCRIPTION OF THE DRAWINGS This invention will be described in greater detail with reference to the drawings in which: Figure 1 depicts a metabolic scheme for the fermentation of lignocellulose hydrolysates.

Figure 2 depicts SEQ ID NO: 1 (the nucleotide sequence of the gene encoding aldose reductase (AR) of S. cerevisiae and flanking sequences), SEQ ID NO: 29 (the deduced amino acid sequence of the AR), and the location and sequence of PCR primers (SEQ ID NOS: 25-28) used to amplify flanking sequences of the invention in Example.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a recombinant vector that can be used to transfer genetic information to a host cell. The recombinant vector includes, as a base molecule a vector known in the genetic engineering (molecular biology) art, including, but not necessarily limited to, a shuttle vector, an expression vector, a cloning (including subcloning) vector, and an integration vector. As used herein, "vector"refers to polynucleotide molecules that can be used to accept, donate, transfer, and/or maintain non-homologous (heterologous) polynucleotide sequences, while a"recombinant vector"according to the invention includes not only a"vector", but heterologous sequences as well. Examples of vectors include, but are not limited to, plasmids, phages or other viruses (including phage and other viral genomes existing independent of an intact virus particle), cosmids, phagemids, and yeast artificial chromosomes (YACs). Preferably, the vector contains all of the sequences necessary for its intended purpose. For example, if it is intended as a long-ter expression vector, it preferably contains an origin of replication that is functional in the host cell. Further, if it is intended as an expression vector (i. e., the base molecule for a recombinant vector), it preferably contains expression controlling sequences (e. g., promoter, enhancer, etc.) operably linked to the sequence which is to be expressed.

Preferred vectors of the invention can comprise, or comprise portions of, a member selected from the group consisting of pUC19, pMA91, pBR322, Ylplac vectors, YEplac vectors, YBplac vectors, and pBluescript vectors. Other preferred vectors are discussed further herein.

The recombinant vector of the invention contains sequences of a gene encoding an aldose reductase (AR), or a related sequence. Preferably, the sequences are flanking sequences taken from one or both ends of the AR- encoding gene. As used hereinafter,"flanking sequence"encompasses any sequence from the AR-encoding gene, but not the entire gene sequence. A flanking sequence is not limited to those sequences at, or near, the termini of the gene. The flanking or flanking-related sequence can be used as a target sequence for not only deleting an endogenous AR-encoding sequence from a host cell, but for subcloning of any desired polynucleotide sequence into the recombinant vector, and preferably into a host cell as well. The AR flanking sequences can be those of any AR-encoding gene known. Preferably, the AR flanking sequences are those of a yeast. More preferably, the flanking sequences are those of the AR-encoding gene of Saccharomyces cerevisiae, or a related sequence. In a highly preferred embodiment, the flanking sequences comprise sequences found within the sequence of SEQ ID NO: 1, or its complementary sequence. The flanking sequences, or sequence related thereto, need not be localized from either end of the sequence of SEQ ID NO: 1, but can be any useful sequence (s) selected therefrom.

For example, two flanking sequences, the first comprising nucleotides 50- 470 and the second comprising nucleotides 1477-1846, of SEQ ID NO: 1 can be used in a vector according to the invention. These sequences can be chemically <BR> <BR> <BR> <BR> synthesized de novo using SEQ ID NO: 1 as a guide or can be amplified in vitro<BR> <BR> <BR> <BR> <BR> <BR> <BR> from SEQ ID NO: 1 using known techniques. It is preferred that the sequences be<BR> <BR> <BR> <BR> <BR> <BR> <BR> amplified from SEQ ID NO: 1 in vitro using known techniques, such as PCR. In this way, primers can be used which have been engineered to contain restriction endonuclease cleavage sites that are not naturally present in SEQID NO: 1.

Incorporation of such cleavage sites aids in subcloning of the amplified flanking sequence into the base vector (e. g., into pUC19). Other modifications to the sequence of SEQ ID N0: 1 can be made as well, as long as the sequence falls within the definition of a related sequences as defined herein.

Other flanking sequence pairs can be chosen, such as: a pair wherein the first flanking sequence comprises nucleotides 1-126 and the second comprises <BR> <BR> <BR> <BR> nucleotides 1639-1814, of SEQ ID NO: 1; a pair wherein the first flanking sequence comprises nucleotides 476-990 and the second comprises nucleotides 1600-1920, <BR> <BR> <BR> <BR> of SEQ ID NO: 1; and a pair wherein the first flanking sequence comprises<BR> <BR> <BR> <BR> <BR> <BR> <BR> nucleotides 191-250 and the second comprises nucleotides 1380-1660, of SEQ ID<BR> <BR> <BR> <BR> <BR> <BR> <BR> NO: 1. As mentioned above, each of flanking sequences preferably contains a restriction endonuclease cleavage site. If a convenient site does not exist in the sequence to be amplified, it can be engineered by incorporation into the primers that are used to amplify the sequence. Preferably the restriction endonuclease cleavage site is unique with respect to the flanking sequences to facilitate subcloning of the amplified sequences into the base vector molecule.

In other preferred embodiments, the sequences are related to the sequence <BR> <BR> <BR> <BR> of SEQ ID NO: 1, or a fragment thereof. The related sequences can be sequences<BR> <BR> <BR> <BR> <BR> <BR> <BR> that hybridize to at least a portion of SEQ ID NO: 1 under stringent conditions. As used herein, stringent conditions are in vitro hybridization conditions in which two polynucleotide molecules are in a substantially hybridized state in the presence of 5XSSPE, 2XDenhardt's solution, and 0.5% (w/v) sodium dodecyl sulfate after at least 20 minutes at 65°C. More stringent conditions are those where the percentage of molecules that are in a hybridized state is lower than the percentage under the above-noted conditions. Such conditions are well-known to the ordinary <BR> <BR> <BR> <BR> artisan, and can include a temperature above 65°C and/or a lower ionic strength solution. By"substantially hybridized state"it is meant that, while not all molecules present that are capable of hybridizing are actually hybridized, enough molecules are found in the hybridized state that hybridized, double-stranded polynucleotide complexes can be detected in a reasonable amount of time using techniques known to the skilled artisan. The length of contiguously hybridized nucleotides is not absolutely critical, nor is the percent identity between the two hybridizing molecules. Rather, it is the combination of these two physical characteristics, among other physical characteristics, that determines which molecules are included within the invention. Such molecules can easily be determined by those of ordinary skill in the art using well-known and widely-practiced techniques.

However, related sequences according to the invention can be characterized in terms of their percent identity with SEQ ID NO: 1, or fragments of SEQ ID NO: 1. In preferred embodiments of this aspect of the invention, the sequences are sequences that show at least 50% identity to at least a portion of <BR> <BR> <BR> <BR> SEQ ID NO: 1. For example, polynucleotide molecules having 50% identity with<BR> <BR> <BR> <BR> <BR> <BR> <BR> SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 are within the present invention. It is preferred that a polynucleotide molecule according to this aspect of the invention <BR> <BR> <BR> have at least 75%, and more preferably at least 85% identity with SEQ ID NO: 1 or a fragment of SEQ ID NO: 1. In highly preferred embodiments, a polynucleotide molecule according to this aspect of the invention has at least 90% identity with <BR> <BR> SEQ ID NO: 1 or a fragment of SEQ ID NO: 1, such as 95%, 98%, or approximately 99% or more.

Preferably, the fragment of SEQ I D NO: 1 comprises at least 10 contiguous <BR> <BR> nucleotides of SEQ ID NO: 1. More preferably, the fragment comprises at least 25,<BR> <BR> and more preferably, at least 50, contiguous nucleotides of SEQ ID NO: 1. In highly preferred embodiments of the invention, the fragment comprises approximately 100 contiguous nucleotides of SEQ ID NO: 1. Other embodiments include fragments having at least 100 contiguous nucleotides, such as approximately 150, 200,300, and 500 contiguous nucleotides.

According to this aspect of the invention, percent identity is calculated using the BLAST sequence analysis program suite, Version 2, available at the NCBI (NIH). All default parameters are used. BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx, all of which are available through the BLAST analysis software suite at the NCBI. These programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (1990,1993) with a few enhancements. Using this publicly available sequence analysis program suite, the skilled artisan can easily identify polynucleotides according to the present invention.

When the flanking sequences are chosen from a sequence of SEQ ID NO: 1, it is preferable that the sequences comprise at least 10 contiguous nucleotides <BR> <BR> from SEQ ID NO: 1. In highly preferred embodiments, the flanking sequences<BR> <BR> comprise at least 25 contiguous nucleotides of SEQ ID NO: 1. In other highly preferred embodiments, the flanking sequences comprise at least 50, and more preferably, approximately 100 or even more contiguous nucleotides of SEQ ID NO:1.

The vectors of the invention can be used for multiple applications. In embodiments of the invention, the vectors are used to specifically delete the AR- encoding gene of a recipient cell. In preferred embodiments, the recipient cell is a yeast cell, such as an S. cerevisiae cell. Other yeasts are well-known to the artisan, and include, but are not limited to, Schizosaccharomyces pombe, Pichia stipitis, and Pichia pastoris. Although the vector of the invention can be used to delete the AR by multiple mechanisms, it is preferred that in vivo deletion of an endogenous AR occur via homologous recombination with the AR-specific or- related sequences present on the vector. That is, by introducing a transfer vector comprising the flanking sequences of a AR, devoid of the entire AR gene, through homologous recombination between the AR sequences on the vector and the endogenous AR-encoding sequences, the endogenous AR-encoding gene can be specifically deleted. As discussed further below, any recombinant strain created in such a way will be unable to metabolize lignocellulose to produce xylitol (or will at the very least have this ability reduced), and may be able to produce a greater quantity of ethanol as a result. This is especially important in the fermentation of lignocellulose by yeasts.

Other techniques for insertion of heterologous nucleic acid molecules into the genome of a recipient cell are known to the skilled artisan in the field. The skilled artisan is free to choose among these techniques to achieve incorporation of the heterologous sequences into the host genome. The choice will be based, at least in part, on the preference of the artisan, as well as the convenience and cost of the technique. For example, the heterologous sequence (s) of one vector construct according to the invention may be inserted into the host genome using homologous recombination via DNA strand crossing over whereas another heterologous sequence may be inserted using the ribosomal integration protocol.

The technique used is not critical to the practice of the invention, and the ability to choose an appropriate technique and protocol is well within the abilities of the skilled artisan in the field.

Of course, specific deletion of the AR-encoding gene of an organism, such as a yeast, can occur simultaneously with the introduction of a new, heterologous sequence or multiple sequences. In essence, the AR-encoding gene is replaced by the new sequence (s), preferably by homologous recombination. By "heterologous"it is meant any sequence that is not identical to the gene being replaced. That is, the heterologous sequence can be an AR-encoding gene from another organism, a mutant form of the AR-encoding gene of the host cell, a gene not known to be related in structure or function to the AR-encoding gene of the host cell, or any other sequence that is not identical to the endogenous host cell gene.

In embodiments where a heterologous sequence is present on the recombinant vector of the invention, the heterologous sequence can be inserted into the recombinant vector using any method known to the skilled artisan. For example, the heterologous sequence can be inserted in the vector using restriction endonuclease cleavage/re-ligation techniques or via homologous recombination.

Preferably, the heterologous sequence is inserted between two flanking sequences of the AR-encoding gene, or between two AR-related sequences such that, upon introduction into the recipient cell, homologous recombination can occur, resulting in incorporation of the heterologous sequence into the genome of the recipient cell.

Further, the heterologous sequence can be generated from multiple different source nucleic acids, or from in vitro amplification of a single or multiple source sequences (e. g., using PCR). The heterologous sequence need not encode a structural protein, but can comprise regulatory elements alone or in combination with a structural gene. The heterologous sequence can also comprise nucleotide sequences with no known function.

In a preferred aspect of the invention, multiple heterologous sequences are inserted between two AR-flanking or-related sequences. In this aspect of the invention, multiple genes can be inserted in place of the AR-encoding gene being deleted. This permits the artisan practicing the invention to modify the genome of the recipient (recombinant) cell such that a naturally-occurring metabolic pathway is shut down while, at the same time, another metabolic pathway can be created or enhanced. According to this aspect of the invention, multiple copies of each of the heterologous sequences can be inserted into the host genome using the vector of the invention. Accordingly, multiple copies of a certain sequence can be included in the recombinant vector of the invention while multiple or single copies of other sequences are included as well. In this way, the expression of all of the genes of a construct can be regulated, broadly or specifically, in respect to each other.

Techniques for manipulating nucleic acids in accordance with this invention are well-known and widely-practiced by the skilled artisan.

For example, a recombinant vector according to the present invention can comprise the xylose isomerase (XI) gene (XyIA) from Thermus thermophilus (Dekker et al.,"Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases", J. Bacteriol. 173 (10): 3078-3083,1991; GenBank Accession No. D90256) inserted between two flanking sequences. A recombinant vector according to the invention can comprise the gene encoding XI from Streptomyces diastaticus inserted between two flanking sequences. Furthermore, a recombinant vector according to the invention can comprise both the gene encoding Xi from T. thermophilus and the gene encoding Xi from S. diastaticus, in any ratio and order.

Other exemplary recombinant vectors can comprise at least one XI-encoding gene and at least one XK-encoding gene (XK). There can be the same number of copies of each gene within the recombinant vector, or different numbers of copies.

Additionally, an XK-encoding gene can be isolated from any known source (e. g., from S. cerevisiae or P. stipitis) and incorporated into a recombinant vector of the invention. Again, multiple heterologous sequences can be present on a single recombinant vector; therefore, the XK-encoding gene, in any copy number, can be incorporated into a recombinant vector comprising an XI-encoding gene, in any copy number.

In preferred embodiments of the invention, the heterologous sequences are coding sequences for enzymes involved in hemicellulose metabolism. It is preferable that these coding sequences encode enzymes involved in the metabolism of hemicellulose to ethanol. Thus, the coding sequences that are inserted into the recombinant vectors of the invention preferably encode enzymes such as XI (encoded by XyIA), XK (encoded by XK and XKS1), XDH (encoded by XYL2), and XR (encoded by XYL1). Other genes that may be included in the recombinant vectors are TAL1 (encoding transaldolase), TKL1 (encoding transketolase), pntA and pnfB (encoding nicotinamide nucleotide transhydrogenase), and cth (encoding transhdrogenase). The nucleotide sequences of representatives of each of these genes are publicly available without restriction.

Many of the nucleotide sequences of the genes encoding heterologous enzymes involved in production of ethanol from xylose are known and publicly available. Using well-known and widely practiced molecular biology techniques (e. g., restriction endonuclease cleavage/re-ligation, PCR, etc.), these sequences, or portions of these sequences, can be subcloned between the flanking sequences of the recombinant vectors of the present invention. Many techniques for subcloning heterologous sequences are known to the skilled artisan. Any suitable technique can be used as long as the technique results in a recombinant vector that can function according to the invention. Where convenient restriction endonuclease cleavage sites are not present in the heterologous sequences, site- directed mutagenesis, and/or mismatches in the primers can be used to engineer such sites prior to subcloning the heterologous sequences into the recombinant vectors of the invention. Such genetic engineering techniques are well within the ordinary artisan's knowledge and abilities, and can be performed without undue or excessive experimentation.

The recombinant vector of the invention can be used to genetically engineer a host cell. Thus, the present invention provides recombinant cells comprising the vector of the invention. The recombinant vector can be maintained within the host cell as an autonomously-replicating molecule, or a portion of the recombinant vector can be incorporated in the genome of the host. Thus, because the recombinant vector of the invention can be an integration vector, the present invention provides recombinant hosts comprising that portion of the recombinant vector which has been integrated into the host cell genome.

In one embodiment of this aspect of the invention, at least a portion of the recombinant vector is integrated into the host genome. In this embodiment, the recombinant cells of the invention have at least one copy of an endogenous AR- encoding gene disrupted or deleted by insertion of sequences of the recombinant vector into the genome. Many of the advantages of the invention are realized through the disruption and/or deletion of a host cell's endogenous AR-encoding gene.

For example, disruption and/or deletion of a gene encoding AR reduces or eliminates the recombinant cell's ability to convert xylose to xylitol. As can be seen from Figure 1, when XR (xylose reductase-an aldose reductase) function is disrupted, direct conversion of xylose to xylitol is blocked, forcing the metabolic degradation of xylose to flow through xylulose. Although it is apparent that xylulose can be converted to xylitol via the XDH, it is also apparent that a greater amount of ethanol is produced from xylose when the AR (XR) is inactive because there is a single pathway leading to xylitol (xylulose to xylitol) instead of two pathways (xylulose to xylitol and xylose to xylitol).

In addition, in embodiments where at least one heterologous sequence is inserted between the AR flanking sequences in the recombinant vector, increased production of xylose-metabolizing enzymes can be effected. For example, the recombinant vector of the invention can comprise one or several copies of an XK- encoding gene inserted between the AR flanking sequences. When this recombinant vector is introduced into a host cell, a recombinant cell according to the invention is achieved, and this recombinant cell comprises not only a non- functional AR-encoding gene, but at least one additional copy of an XK-encoding gene. Upon expression, the recombinant cell will produce increased amounts of XK, thus increasing the relative amount of xylulose converted to xylulose-5- phosphate (relative to the amount of xylulose converted to xylitol).

As discussed above, the recombinant vectors of the invention can contain multiple copies of any number of genes involved in xylose metabolism, and especially xylose conversion to ethanol. Thus, in addition to containing at least one copy of an XK-encoding gene, the recombinant vector can also contain at least one copy of any of the genes encoding enzymes of the Embden-Meyerhof-Parnas pathway or other enzymes necessary for conversion of glucose to ethanol in yeasts. Such enzymes include, but are not necessarily limited to, glyceraldehyde- 3-phosphate dehydrogenase, 3-phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, pyruvate decarboxylase, and alcohol dehydrogenase. By supplying copies of genes encoding these enzymes, conversion of xylose to ethanol can be improved to an even greater degree. This is because the increased number of ethanol-specific enzymes will permit the recombinant cell to rapidly utilize any xylulose-5-phosphate present. By rapidly depleting the xylulose-5-phosphate, and maintaining a very low level of it, the amount of xylitol produced from xylulose will be substantially decreased because rapid production and subsequent consumption of xylulose-5-phosphate will diminish the amount of xylulose available for conversion to xylitol.

Thus, the recombinant cells of the invention provide a solution to the need in the art for increased production, at economical rates, of ethanol from compositions comprising lignocellulose and its hydrolysates. Although the recombinant cells of the invention find their most highly valued use in industrial settings, they can also be utilized as research tools and as basic materials for further recombinant manipulation. Furthermore, although the above discussion is focused on recombinant yeast cells, the recombinant cells are not limited to such cells.

Rather, any cell comprising an AR-encoding gene is encompassed by this invention.

Accordingly, the invention provides a method of making recombinant cells.

The method comprises providing a recombinant vector according to the invention and inserting that vector into a host cell to create a recombinant cell. The method can optionally include culturing the recombinant cell under conditions where the heterologous sequences present on the recombinant vector are expressed. The method can also optionally include maintaining the recombinant cell under conditions where integration of at least a portion of the recombinant vector into the host genome occurs. Furthermore, the method can optionally include storing the recombinant cells in an environment where the cell remains viable and stable for extended periods of time (e. g., frozen or lyophilized). Conditions under which these optional method steps can be performed are well-known and widely practiced by the skilled artisan.

Suitable host cells include any prokaryotic and eukaryotic cells having an AR-encoding gene. In preferred embodiments of the invention, the host cell is a yeast, especially a yeast of the Saccharomyces family. Exemplary host cells <BR> <BR> <BR> <BR> include CEN. PK (pgi6), CEN. PK (pgi7), isolate 3 of Lindén et al., 1992, and isolate 10 of Lindén et al., 1992.

In addition, the invention provides a method of degrading lignocellulose- containing compositions and compositions containing lignocellulose hydrolysates.

The method comprises combining at least one recombinant cell of the invention with the lignocellulose-containing composition and incubating the mixture under conditions that permit degradation of the lignocellulose or other components present in the composition.

Preferably, the method is used as a method of producing ethanol from lignocellulose-containing compositions and compositions comprising lignocellulose hydrolysates. The method can be a method of fermenting lignocellulose or lignocellulose hydrolysates, and provides the production of relative high levels of ethanol and relatively low levels of xylitol. The method of producing ethanol comprises the following steps: (a) providing a composition containing lignocellulose or a hydrolysate or metabolic degradation product of lignocellulose, (b) providing a recombinant cell according to the invention, (c) combining the recombinant cell of the invention with the composition containing lignocellulose or a hydrolysate or metabolic degradation product of lignocellulose, (d) allowing the recombinant strain to metabolize the lignocellulose or breakdown product, and (e) harvesting or purifying the metabolic products produced by the breakdown of the lignocellulose or hydrolysate.

Preferably, the method results in production of ethanol. In this situation, the method optionally further includes purifying or isolating the ethanol from other components present in the mixture formed by the combination of the recombinant cell and the composition comprising lignocellulose or its hydrolysates. The method of producing ethanol provides high levels of ethanol production, and low levels of xylitol production from lignocellulose-containing compositions, especially those in which xylose is found. The amount of ethanol produced from lignocellulose- containing compositions or lignocellulose hydrolysate-containing compositions is much greater than the amount that can be produced from lignocellulose-or lignocellulose hydrolysate-degrading strains currently available.

It is highly preferable that the conditions under which the recombinant strain is allowed to metabolize or degrade components of the composition include anaerobic conditions; however, pseudo-anaerobic conditions are also preferred while aerobic conditions are acceptable.

The recombinant strains of the invention are useful for the efficient fermentation of lignocellulose hydrolysates.

EXAMPLES Example 1: Amplification of the IA gene from Thermus thermophilus and construction of plasmid pBXI Two primers were used to amplify the xylA gene using PCR amplification: 5'-GCGCTGATCATCTAGAATGTACGAGCCCAAACCGGAGCACAG-3' (SEQ ID NO: 2; 5'primer) and 5'-GCTTTGATCATCTAGATCACCCCCGCACCCCCAGGAGGTACT-3' (SEQ ID NO: 3; 3'primer).

Both primers contained restriction endonuclease sites for Bcll and Xbal.

The PCR mixture contained: PCR buffer with 2 mM MgSO4,0.8 mM dNTPs, 0.3uM of each primer, 0.2 pg template (pUC19-XI), and 2.5 units Pwo DNA polymerase (Boehringer Mannheim).

A DNA Thermal Cycler (Perkin Elmer Cetus) was used for amplification of the gene under the following conditions: melting temperature 94°C (1 min), annealing temperature 58°C (1 min), and polymerization temperature 72°C (1 min). Twenty-eight cycles were run with a subsequent polymerization period of 7 min at 72°C. The amplified DNA fragment was digested with Bcll and ligated into the Bglll site of pMA91, resulting in plasmid pBXI. The Bglll site of pMA91 was placed between the phosphoglycerate kinase (PGK1) promoter and terminator.

Example 2: Amplifiation of the XYL 1 gene of P. stipitis and creation of plasmid UA103 Two primers were used to amplify the XYL1 gene from P. stipitis using PCR: 5'-GCGGATCCTCTAGAATGCGTTCTATTAAGTTGAACTCTGG-3' (5' primer; SEQ ID NO: 4), and 5'-TTGGATCCTCTAGATTAGACGAAGATAGGAATCTTGTCCC-3' (3' primer; SEQ ID NO: 5).

The primers contained BamHl and Xbal restriction sites at both ends. PCR was performed as in Example 1. The PCR amplified chromosomal region was cut with BamHl and subcloned into the Bglll site of the yeast expression vector pMA91 between the PGK promoter and terminator, giving the plasmid pUA103.

Example 3: Amplification of the XYL1 and XYL2 genes of P. stipitis Plasmids comprising the XYL1 and XYL2 genes of P. stipitis (pMW103 and pMW104, respectively; Walfridsson et al, 1995) were used to amplify the XYL1 and XYL2 genes using the following primers: 5'-CGCAGGATCCACTAGAATGCCTTCTAT-3' (SEQ ID NO: 6) 5'-TCCTCTAGATTGGACGAAGATAGGAAT-3' (SEQ ID NO: 7) 5'-GCGTCTAGAATGACTGCTAACCCTTCC-3' (SEQ ID NO: 8) <BR> <BR> 5'-GCGCGAAGCTTAGATCTTTACTCAGGGCCGTCAA-3' (SEQ ID N0: 9)<BR> <BR> 5'-GCCTCTAGAATGCCTTTATTAAG-3' (SEQ ID N0: 10) 5'-GCGCGAAGCTTGGATCCTTAGACGAAGATAGGAA-3' (SEQ ID NO: 11) 5'-CGCAGGATCCACTAGAATGACTGCTAACCCTTC-3' (SEQ ID NO: 12) 5'-TCCTCTAGAACCCTCAGGGCCGTCAATG-3' (SEQ ID NO: 13) 5'-GCCTCTAGACCATCTCCAACCGCTAGCA CTAACCAAATGCCTTCTATTAAGTTG-3' (SEQ ID NO: 14).

SEQ ID NO: 6 corresponds to the 5'end of the XYL1 gene. SEQ ID NO: 7 corresponds to the 3'end of the XYL1 gene. The XYL1 gene was amplified by PCR from plasmid pMW103. The amplified DNA was then cleaved with BamHl and Xbal, and inserted into pUC19.

SEQ ID NO: 8 corresponds to the 5'end of the XYL2 gene. SEQ ID NO: 9 corresponds to the 3'end of the XYL2 gene. The XYL2 gene was amplified by <BR> <BR> PCR from plasmid pMW104. The amplified DNA was then cleaved with Hindlll and Xbal, and inserted into pUC19. The digested amplified DNA was also inserted into the pUC19 derivative disclosed above, which contained the amplified XYL1 gene.

Example 4: Amplification of the pnM and pntB from E. coli The pnM gene was amplified from E. coli using PCR and the following primers: 5'-GCGCGAGATCTTCTAGAATGCGAATTGGCATACCAAG-3' (SEQ ID NO: 15) 5'-CGCGCAGATCTTCTAGATTAATTTTTGCGGAACATTTTC-3' (SEQ ID NO: 16) The pntB gene was amplified from E. coli using PCR and the following primers: 5'-GCGCGAGATCTAAAATGTCTGGAGGATTAGTTAC-3' (SEQ ID NO: 17) 5'-CGCGCAGATCTTTACAGAGCTTTCAGGATTGC-3' (SEQ ID NO: 18).

The amplified genes were digested with the appropriate restriction endonucleases and subcloned into pUC19 vectors, alone and in combination.

Example 5: Amplification and subcloning of the cth gene of Azotobacter vinelandii The cth gene of A. vinelandii, encoding the transhydrogenase, was amplified by PCR using the following primers: 5'-GtnTA (C/T) AA (C/T) TA (C/T) GA (C/T) GTnGTnGTnAT (A/C/T)-3' (SEQ ID NO: 19) 5'- (A/G) TA (A/G) TT (A/G) AAnGTnGT (A/G) TT (A/T/G) AT (A/G) AA (A/G) TA-3' (SEQ ID NO: 20).

From the PCR reaction, a fragment of 1300 bp was obtained and subcloned into pUC18.

Example 6: Amplification of P. stipitis polyol dehydrogenase gene The gene encoding polyol dehydrogenase was amplified from P. stipitis using PCR and the following primers: 5'-GGATCCAGATCTATGGACTACTCATACGCT-3' (SEQ ID NO: 21) 5'-GGATCCAGATCTTTAAACTGTGGGTCGTAT-3' (SEQ ID NO: 22) The gene was amplified and subcloned into pUC18.

Example 7: Cloning the xylulose kinase gene from S. cerevisiae The XK from S. cerevisiae was amplified and subcloned using PCR and the followingprimers: 5'-GCGGATCCTCTAGAATGGTTTGTTCAGTAATTCAG-3' (SEQ ID NO: 23) 5'-AGATCTGGATCCTTAGATGAGAGTCTTTTCCAG-3' (SEQ ID N0: 24).

Example 8: Amplification of AR flanking sequence Two flanking sequences from SEQ ID NO: 1 were amplified using PCR and the following primers: Upstream flanking region: 5'-GATCGAATTCTTTGTAACTGTAATTTCACTCATGC-3' (SEQ ID NO: 25; corresponding to nucleotides 50-74 of SEQ ID NO: 1, with an EcoRl site engineered at the 5'end of the primer), and 5'-GTACAAGCTTTTTCCAATTTTCCTTTACGATTT-3' (SEQ ID NO: 26; complementary to nucleotides 470-448 of SEQ ID NO: 1, with a Hindlll site engineered at the 5'end of the primer); Downstream flanking region: 5'-GATCAAGCTTAATCCATACTCAACGACGATATG-3' (SEQ ID NO: 27; corresponding to nucleotides 1477-1499 of SEQ ID NO: 1, with a Hindlll site engineered at the 5'end of the primer), and 5'-GTACGGATCCGTCGCTCATATCTTGCTGTGT-3' (SEQ ID NO: 28; complementary to nucleotides 1846-1826 of SEQ ID NO: 1, with a BamHl site engineered at the 5'end of the primer).

The primers were used to amplify regions of SEQ ID NO: 1 and insert convenient restriction endonuclease cleavage sites into the sequences to facilitate subcloning of the flanking sequences into the base vector molecule.

Example 9: Construction of plasmid pUSM1004 Using PCR and primers based on SEQ ID NO: 1, flanking sequences were amplified and subcloned into base vector pBR322. The 3'flanking region was ligated between the Hindlll and BamHl sites in pBR322 to create plasmid pUSM1002. The 5'flanking region was then ligated between the Hindlil and EcoRl sites of pUSM1002 to create plasmid pUSM1004, which contained both a 5' flanking sequence and a 3'flanking sequence of the AR-encoding gene of S. cerevisiae.

Example 10: Construction of plasmid pUSM1006 Plasmid pUSM1004 from Example 9 was digested with BamHl and EcoRI to release the AR flanking sequences. The liberated BamHl/EcoRl fragment was subcloned into base shuttle vector pUC8+URA to give plasmid pUSM1006.

Example 11: Construction of recombinant strains YUSM1006, MATa and cr Plasmid pUSM1006 was transfected into yeast strain CEN. PK2-1C and CEN. PK2-1 D using standard techniques. Transfectants were cultured to permit integration of the vector into the host genome. Stable recombinant cells were obtained.

Example 12: Construction of recombinant strain YKTR101 The xylA and XK genes were subcloned into plasmid pUSM1006.

The resulting plasmid was transfected into yeast strain using standard techniques. Transfectants were cultured to permit integration of the vector into the host genome. Stable recombinant cells (YKTR101) were obtained having the xylA and XK genes integrated into the host genome.

The invention has been described in detail above with reference to preferred embodiments. However, it will be understood by the ordinary artisan that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. All references cited herein are hereby incorporated by reference in their entirety.