PLANT XYLOSE ISOMERASE

Abstract: The present invention relates to a neucleic acid sequence which codes upon expression in a prokaryotic or eucaryotic host cell for a polypeptide having xylose isomerase activity, which nucleic acid sequence is selected from: a) the nucleic acid sequences shown in SEQ. ID. No. 1 and SEQ. ID. No. 2 or the complementary strands thereof; b) nucleic acid sequences which hybridise to the sequences defined in (a) above; c) nucleic acid sequences which, but for the degeneracy of the genetic code, would hybridise to the sequences defined in (a) or (b) above and which code for the same polypeptides as those defined in (a) or (b) above; and to a process of producing ethanol from lignocellulosic materials comprising contacting cells that express such nucleic acid sequences.

Description: Plant xylose isomerase This invention relates to nucleic acid sequences encoding a novel plant xylose isomerase, to vectors carrying the sequen¬ ces and to the use of the isomerase in a process of ethanol production. D-xylose is a five-carbon sugar. It is present in nature, for example as xylan polymer in plant hemicellulose. As a carbon source, D-xylose has been reported to be utilized widely by bacteria and to a lesser extent by fungi. D-xylose is first converted to its eto isomer, D-xylulose, which is then phosphorylated to D-xylulose-5-phosphate, a normal pentose phosphate cycle intermediate. There are two possible routes for the isomerization of D-xy¬ lose: (a) direct conversion to D-xylulose by an isomerase; or (b) an oxidoreductive pathway, in which xylitol is an interme¬ diate, and coenzymes for the two oxidoreductases involved are needed. The oxidoreductive pathway has been reported to be the main route in fungi. Xylose isomerase catalyses the direct isomerization of D-xy¬ lose to D-xylulose, and vice versa, with the direction of the reaction dependent on the relative concentrations of the aldo and keto forms and the reaction conditions. Xylose isomerase also catalyses the interconversion of the 5-carbon sugars D-ribose and D-ribulose and the interconversion of the 6-car- bon aldose sugar, D-glucose, and its keto isomer, D-fructose. Thus, bacterial xylose isomerases are used industrially to produce D-fructose from D-glucose. Some eucaryotes have also been found to be capable of convert¬ ing xylose to xylulose, for example certain yeasts, such as Pichia stipitis. However, the oxidoreductive pathway men¬ tioned above is at least partially responsible for these observations because it has not always been possible to prove the existence of a xylose isomerase and because oxidoreductive enzymes have been isolated from Pichia stipitis and the other yeasts that have been found to be able to metabolise xylose. In plants, certain tissues are believed to contain xylose isomerase because partially purified extracts of corn pollen and wheat germ are capable of converting xylose to xylulose. This conversion occurs in the absence of NADPH and is not known to require any coenzymes, though Mn 2 * or Mg=* ions are required. In order to produce ethanol by fermentation of lignocellulosic material, it is desirable that the organism responsible for the fermentation is able to convert xylose to xylulose. Such organisms generate a high yield of ethanol because they are able to degrade hemicellulosic xylose derived from xylan polymers in lignocellulosic materials. The principal distill¬ ers' yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe are not naturally able to iso erise D-xylose to D-xylu¬ lose although they readily ferment D-xylulose to ethanol. Some attempts have been made to overcome this problem by transforming S. cerevisiae and S. pombe with bacterial xylose isomerases or oxidoreductive enzymes from yeasts that natural¬ ly utilise D-xylose. Also, attempts have been made to enable S. cerevisiae and S. pombe to isomerise D-xylose to D-xylulose by adding bacterial isomerases to fermentation broths. Yeasts transformed with bacterial isomerases do not produce ethanol efficiently, despite the fact that bacterial xylose isomerases have higher specificity for xylose than for glucose or ribose. It is believed that bacterial isomerase enzymes are poorly expressed in yeast or that they aggregate or are cleaved proteolytically, owing to differences between the procaryotic cells from which they are derived and the eucaryo- tic yeast host cells. A eucaryotic xylose isomerase has now been isolated. This is the barley xylose isomerase, which is the first eucaryotic xylose isomerase to be isolated and therefore the first plant xylose isomerase to be isolated. It is to be expected that the eucaryotic barley enzyme will be more efficiently ex¬ pressed in yeast than bacterial enzymes because of the genetic similarities between the eucaryotic plant cell from which the enzyme is derived and the eucaryotic yeast cell in which it is expressed. The amino acid sequence of the enzyme has been determined, the gene encoding it has been cloned and its genomic and cDNA sequences have been determined. Further, it has been found that this barley xylose isomerase has a very high specificity towards D-xylose, as compared to D-glucose and D-ribose. Indeed, in experiments conducted by the present inventors, the barley enzyme was not observed to have any glucose isomerase or ribose isomerase activity. Owing to this high specificity, the enzyme can be used specifically to produce xylulose from xylose, even in the presence of other sugars such as glucose and ribose, as the barley enzyme will not isomerise these other sugars. Accordingly, distillers' yeasts that express the barley xylose isomerase can be used to ensure increased yields of ethanol from xylose in lignocellulosic materials, even in the presence of glucose. Also, the enzyme and cells expressing it, can be used in the modification of xylans. Accordingly, the present invention provides: an isolated nucleic acid sequence which codes upon expression in a procaryotic or eucaryotic host cell for a polypeptide having xylose isomerase activity, which nucleic acid sequence is selected from: a) the nucleic acid sequences shown in SEQ. ID. No. 1 and SEQ. ID. No. 2 or the complementary strands thereof; b) nucleic acid sequences which hybridise to the sequences defined in (a) above; c) nucleic acid sequences which, but for the degeneracy of the genetic code, would hybridise to the sequences defined in (a) or (b) above and which code for the same polypeptides as those defined in (a) or (b) above; a polypeptide encoded by a nucleic acid sequence as defined above; a vector comprising a nucleic acid sequence as defined above; cells transformed or transfected with a vector such a vector; a process of producing ethanol which comprises: (i) contacting such transformed cells with a substrate that comprises one or more carbon sources selected from xylose and polymerised xylose moieties; (ii) culturing the said cells in conditions under which the isomerisation of D-xylose to D-xylulose occurs and under which the D-xylulose is further catabolized to ethanol; and (iii) recovering the ethanol; an antibody to a polypeptide as defined above; a process of producing a polypeptide as defined above compris¬ ing expressing a nucleic acid sequence as defined above in a cell as defined above. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Xylose isomerase activity in barley malted for various lengths of time (Days). Figure 2. Effect of temperature on enzyme activity. Figure 3. Effect of pH on enzyme activity. Figure 4. Exon/intron structure of the barley xylose isome¬ rase gene. Figure 5. The exon/intron junctions of the barley xylose isomerase gene. Figure 6. Construction of plasmid pALK710, containing the full lenghth xylose isomerase cDNA. Figure 7. Construction of plasmids pALK720 and pALK721. Figure 8. Construction of plasmids pALK724 and pALK725. Figure 9A. The 5' -terminal end of the cDNA sequence of the full cDNA sequence of barley xylose isomerase disclosed in SEQ. ID. No. 2 and the corresponding part of the full protein sequence of barley xylose isomerase. The total amino acid sequence is dis¬ closed in SEQ. ID. No. 3 and Figures 9A and 9B. Figure 9B. The 3' -terminal end of the cDNA sequence of the full cDNA sequence of barley xylose isomerase disclosed in SEQ. ID. No. 2 and the corresponding part of the full protein sequence of barley xylose isomerase. The total amino acid sequence is disclosed in SEQ. ID. No. 3 and Figures 9A and 9B. The nucleic acid sequences of the present invention are preferably DNA, though they may be RNA. It will be obvious to those of skill in the art that, in RNA sequences according to the invention, the U residues shown in SEQ. ID. No. 1-5 will be replaced by T. The nucleic acid sequences of the present invention are not limited to the sequences of SEQ. ID. No. 1 and SEQ. ID. No. 2. Rather, the sequences of the invention include sequences that are closely related to these sequences and that encode a polypeptide having xylose isomerase activity, xylose isomerase activity being an ability to catalyse the direct interconver¬ sion of xylose and xylulose. These sequences may be prepared by altering those of SEQ. ID. No. 1 or 2 by any conventional method, or isolated from any organism or made synthetically. Such alterations, isolations or syntheses may be performed by any suitable method, for example by the methods of Sambrook et al. : (Molecular Cloning: A Laboratory Manual; 1989). For example, the sequences of the invention include sequences that are capable of selective hybridisation to those of SEQ. ID. No. 1 and/or SEQ. ID. No. 2 and that encode a polypeptide having xylose isomerase activity. Such sequences capable of selectively hybridizing to the DNA of SEQ. ID. No. 1 and/or 2 will be generally at least 70 %, preferably at least 80 or 90 % and more preferably at least 95 % homologous to the DNA of SEQ. ID. No. 1 or 2 over a region of at least 20, preferab¬ ly at least 50, for instance 100, 500 or 1000 or more con¬ tiguous nucleotides. Such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al. (1989): Molecular Cloning : A Laboratory Manual). For example, if high stringency is required, suitable conditions include 0.2 x SSC at 60 " C. If lower stringency is required, suitable conditions include 2 x SSC at 60 ' C. Also included within the scope of the invention are sequences that differ from those defined above because of the degeneracy of the genetic code and encode the same polypeptide having xylose isomerase activity, namely the polypeptide of SEQ. ID. No. 3, or a polypeptide related to it in any of the ways defined below. In particular, the non-coding portions of nucleic acid sequenc¬ es of the invention including the introns of SEQ. ID. No. 1, may be modified in any way that does not destroy the xylose isomerase activity of the encoded polypeptide. The nucleic sequences of the invention may be of any length as long as they encode a peptide having xylose isomerase activi¬ ty. For instance, a nucleic acid sequence according to the invention will typically comprise the parts of the native gene sequence that encode the active site of the native protein. A nucleic acid sequence according to the invention may be a contiguous fragment of the native sequence or a sequence that is related to it in any of the ways described above. Alterna¬ tively, nucleic acid sequences of the invention may comprise DNA sequences that are not contiguous in the native sequence. These sequences may be fragments of the native DNA sequence or nucleic acid sequences that are related to such fragments in any of the ways described above. Nucleic acid sequences of the invention will preferably comprise at least 50 bases, for example 50 to 100, 100. to 500, 500 to 1000, or 1000 to 2000 bases. Similarly, the polypeptides of the invention are not limited to the polypeptide of SEQ. ID. No. 3. Rather, the polypep¬ tides of the invention also include polypeptides with sequenc¬ es closely related to that of SEQ. ID. No. 3 that have xylose isomerase activity. These sequences may be prepared by altering those of SEQ. ID. No. 3 by any conventional method, or isolated from any organism or made synthetically. Such alterations, isolations or syntheses may be performed by any conventional method, for example by the methods of Sambrook e_t al. (Molecular cloning: A Laboratory Manual; 1989). In partic¬ ular, polypeptides related to that of SEQ. ID. No. 3 may be prepared by modifying DNA sequences as shown in SEQ. ID. No. 1 or 2 and expressing them recombinantly. Polypeptides of the invention may include subs itutions, deletions, insertions, or extensions that distinguish them from SEQ. ID. NO. 3 as long as these do not destroy the xylose isomerase activity of the polypeptide. A substitution, deletion or insertion may suitably involve one or more amino acids, typically from one to five, one to ten or one to twenty amino acids. For example, a substitution, deletion or insertion of one, two, three, four, five, eight, ten, fifteen, or twenty amino acids. Typically, a peptide of the invention has at least 40 %, at least 60 %, at least 80 %, at least 90 %, or at least 95 % sequence identity to native barley xylose isomerase sequence (SEQ. ID. NO. 3. ). In general, the physicochemical nature of the sequence of SEQ. ID. No. 3 should be preserved in a sequence of the invention. Such sequences will generally be similar in charge, hydropho- bicity and size to that of SEQ. ID. No. 3. Examples of substitutions that do not greatly affect the physicochemical nature of amino acid sequences are those in which an amino acid from one of the following groups is substituted by a different amino acid from the same group: H, R and K I, L, V and M A, G, S and T D, E, P and N. As far as extensions are concerned, a sequence of one or more amino acids may be provided at either or both of the C- and N- termini of the sequence of SEQ. ID. No. 3 or a sequence related to it in any of the ways defined herein. An extension may comprise up to 5, up to 10, up to 20, up to 50, or up to 100 amino acids. For example, an extension may comprise one, two, three, four, five or ten amino acids. A polypeptide of the invention may be subjected to one or more chemical modifications, such as glycosylation, sulphation, COOH-amidation or acylation. A polypeptide of the invention may comprise multiple copies of the sequence of SEQ. ID. NO. 3, or a sequence related to it in any of the ways defined herein. A polypeptide of the invention may be of any length as long as it has xylose isomerase activity. For instance, a polypeptide of the invention might comprise the active site of the native protein and therefore have xylose isomerase activity despite being much smaller than the native protein. Polypeptides according to the invention may be composed of a contiguous fragment of the native protein sequence or a fragment that is related to it in any of the ways described above. Alternative¬ ly, polypeptides of the invention may comprise amino acid sequences that are not contiguous in the native protein. These amino acid sequences may be identical to parts of the native amino acid sequence or related to such parts in any of the ways described above. Polypeptides according to the invention preferably comprise at least 10 amino acids, for example 10 to 20, 20 to 50, 50 to 100, 100 to 200, or 200 to 500 amino acids. Polypeptides according to the invention may be purified or substantially purified. Such a polypeptide in substantially purified form will generally comprise the polypeptide in a preparation in which more than 90 %, eg. 95 %, 98 % or 99 % of the peptide material in the preparation is that of a poly¬ peptide or polypeptides according to the invention. The nucleic acid sequences and polypeptides of the invention were originally derived from the barley genome. However, nucleic acid sequences and/or polypeptides of the invention may also be obtained from other eucaryotic genomes, especially other plant genomes. They may be obtained either by conven¬ tional cloning techniques or by probing genomic or cDNA libraries with nucleic acid sequences according to the inven¬ tion. This can be done by any conventional method, such as the methods of Sambrook et al. (Molecular Cloning: A Labora¬ tory Manual; 1989). The polypeptides of the invention may be linked to a signal sequence capable of directing their secretion from host cells, such as yeast cells, for example cells of S. cerevisiae or S. pombe. Accordingly, nucleic acid sequences, of the invention may encode such signal sequences in addition to polypeptide sequences having xylose isomerase activity. The nucleic acid sequence encoding the signal sequence must be positioned relative to the sequence encoding the polypeptide having xylose isomerase activity in such a way that the signal sequence is expressed in the host cell and is capable of directing secretion of the polypeptide having xylose isomerase activity. Typically, the nucleic acid encoding the signal sequence will be 5' to that encoding the polypeptide having xylose isomerase activity. The nucleic acid encoding the signal sequence may be immediately 5' to that encoding the polypeptide having xylose isomerase activity such that, when the polypeptide is expressed, the signal sequence is immediate¬ ly N-terminal to the polypeptide having xylose isomerase activity. Alternatively, there may be intervening sequence between the nucleic acid encoding the signal sequence and that encoding the polypeptide having xylose isomerase activity. Further, the nucleic acid encoding the signal sequence must be in the same reading frame as that encoding the polypeptide having xylose isomerase activity. Preferred signal sequences include the Hormoconis resinae glycoamylose signal sequence, though any signal sequence capable of directing secretion of the polypeptide having xylose isomerase activity may be used. A nucleic acid sequence according to the invention may be included within a vector, suitably a replicable vector, for instance a replicable expression vector. Such an expression vector comprises an origin of replication so that the vector can be replicated in a host cell such as a bacterial host cell or a yeast host cell. A suitable vector will also typically comprise the following elements, usually in a 5' to 3' arrangement: a promoter for the directing expression of the nucleic acid sequence and optionally a regulator of the promoter, a translational start codon, a nucleic acid sequence according to the invention encoding a polypeptide having xylose isomerase activity. The vector may also contain one or more selectable marker genes, for example an ampicillin resistance gene for the identification of bacterial transformants or a marker gene that allows selection of yeast transformants. Optionally, the vector may also comprise an enhancer for the promoter. The vector may also comprise a polyadenylation signal operably linked 3' to the nucleic acid encoding the functional protein. The vector may also comprise a transcriptional terminator 3' to the sequence encoding the polypeptide of the invention. The vector may also comprise one or more introns or other coding sequences 3' to the sequence encoding the polypeptide having xylose isomerase activity. The intron or introns may be from barley (the organism from which the sequences of the invention are derived) or the host organism which is to be transformed with the vector or from another eucaryotic organ¬ ism. In an expression vector, the nucleic acid sequence of the invention is operably linked to a promoter capable of express¬ ing the sequence. "Operably linked" refers to a juxtaposition wherein the promoter and the nucleic acid sequence encoding the polypeptide having xylose isomerase activity are in a relationship permitting the coding sequence to be expressed under the control of the promoter. Thus, there may be ele¬ ments such as 5' non-coding sequence between the promoter and coding sequence. These elements may be native either to barley or to the organism from which the promoter sequence is de¬ rived. Alternatively, the said element or elements may be native to neither the organism from which the promoter sequence is derived nor to barley. Such sequences can be included in the construct if they enhance or do not impair the correct control of the coding sequence by the promoter. The expression vector may be of any type. The vector may be in linear or circular form. For example, the construct may be incorporated into a plasmid vector. Those of skill in the art will be able to prepare suitable vectors comprising nucleic acid sequences encoding polypeptides having xylose isomerase activity starting with widely available vectors which will be modified by genetic engineering techniques such as those described by Sambrook et al. (Molecular Cloning: A Laboratory Manual; 1989). So far as plasmid vectors are concerned, two suitable starting vectors are the plasmids PAAH5 (Ammerer (1983): Meth. Enzymol 101, 192-201) and PEVPU (Hildebrandt et al. (1989): FEBS 243 (2), 137-140), which are widely avail¬ able. In an expression vector, any promoter capable of directing expression of a sequence of the invention may be operably linked to the nucleic acid, sequence of the invention. Particu¬ larly suitable promoters are yeast promoters, for example promoters derived from Kluveromyces spp, Saccharo yces cerevi¬ siae or Schizosaccharomyces pombe. Suitable promoters for the expression of the heterologous genes, such as the barley isomerase gene of the invention, in yeast may be constitutive or regulable. Examples of suitable constitutive promoters are the PDC, PGK, GAPDH, TRP1 and MFαl promoters. Suitable regulable promoters include the PH05, ADH1, CUP1, GAL 1, GAL10 and PRB1 promoters. Promoters from viral genes that are expressed in eucaryotic host cells are also suitable. A particularly preferred promoter is the yeast alcohol dehydroge- nase (ADH1) promoter. Typically, nucleic acid sequences according to the invention will be inserted into such vectors in a sense orientation. However, nucleic acid sequences according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense R A may also be produced by synthetic means. Such antisense RΝA may be used in a method of controlling the levels of the protein of SEQ. ID. No. 3 or a protein encoded by a related nucleic acid sequence in a cell. Vectors according to the invention may be used in vitro, for example for the production of RNA hybridisable to the cDNA. Such vectors may be used to transfeet or transform a host cell. Depending on the type of vector, they may be used as cloning vectors to amplify DNA sequences according to the invention or to express this DNA in a host cell. A further embodiment of the invention provides host cells transformed or transfected with the vectors for the replica¬ tion and/or expression of nucleic acid sequences according to the invention, including the DNA SEQ. ID. No. 1 or SEQ. ID. No. 2. The cells will be chosen to be compatible with the vector and may for example be bacterial cells or yeast cells. Transformed or transfected bacterial cells for example E. coli cells, will be particularly useful for amplifying nucleic acid sequences of the invention. Transformed or transfected yeast cells are particularly preferred for expression of polypeptides according to the invention, which allows them to convert xylose to xylulose. Preferred species of yeast include the distillers' yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. A particularly preferred strain of S. cerevisiae YF135 is strain ALK0246, with the scientific description α leu 2-3, leu 2-112, his 3-11, his 3-15, which strain was deposited under the Budapest Treaty at the Centraalbureau voor Schimmelcultures (CBS) in the Netherlands under deposit number CBS 601.94 on 7th December 1994. A particularly preferred strain of S. pombe is strain ALKO 2185, the scientific description of which is leu 1.32 h~, which strain was deposited under the Budapest Treaty at the Centraalbureau voor Schimmelcultures (CBS) in the Netherlands under deposit number CBS 602.94 on 7 December 1994. The preferred cultivation conditions for the deposited strains are 2 days in a temperature of 30 "C in for example GYPA- or GPYA-medium. Viability tests are performed in the same cultivation condition preferably in GPYA-medium. The strains are preferably stored as freeze dried or in frozen stare at -70 'C or lower temperatures. The cells may be transformed or transfected by any suitable method, such as the methods disclosed by Sambrook et al. (Molecular cloning: A Laboratory Manual; 1989). For example, vectors comprising nucleic acid sequences according to the invention may be packaged into infectious viral particles, such as example retroviral particles. The constructs may also be introduced by electroporation, calcium phosphate precipita¬ tion, biolistic methods or by contacting naked nucleic acid vectors with the cells in solution. In the said nucleic acid vectors with which the host cells are transformed or transfected, the nucleic acid may be DNA or RNA, preferably DNA. The vectors with which the host cells are transformed or transfected may be of any suitable type. For example the vectors may be able to effect integration of nucleic acid sequences of the invention into the host cell genome or they may remain free in the host cell. Typically, the vectors will be expression vectors, such as a retroviral vector or a DNA expression vector as defined herein. For example, the vector used for transformation construct may be a plasmid vector as defined herein. The transformed or transfected cells of the invention can be used in a process of production of ethanol production. Cells according to the invention that express polypeptides having xylose isomerase will be capable of converting xylose to xylulose, which is then fermented to ethanol. Such a process of producing ethanol will typically comprise contacting one or more suitable substrates with transformed or transfected cells according to the invention. The thus produced ethanol will typically be recovered by any method known in the art, such as distillation. Suitable substrates for ethanol production include xylose itself and any compound that the cells can convert into xylose. For example, suitable, substrates include polymers that contain xylose moieties. Thus, xylan and xyloglucan polymers, which comprise xylose moieties, are suitable sub¬ strates. Accordingly, hemicellulosic and lignocellulosic substrates, such as plant biomass, are suitable substrates. In processes of ethanol production according to the present invention, xylose is typically released from xylan by enzymat¬ ic or chemical hydrolysis under acidic or basic conditions, or by heating or by a combination of these techniques. For example xylose can be released from xylan by a combination of acidic or basic hydrolysis and heating, or by heating under pressure. The yeasts typically used in ethanol production are not capable of hydrolysing xylans enzymatically to release xylose although some yeasts are capable of doing so and of metabolising the xylose to ethanol. Such yeasts, when trans¬ formed with DNA according to the invention, are included within the scope of the invention. Although the preferred processes of ethanol production accord¬ ing to the invention comprise contacting transformed or transfected cells with a suitable substrate, other processes of producing ethanol are also possible. For example, a polypeptide according to the invention may be added to a cellular fermentation broth in order to liberate xylose outside the cells, which is then taken up and metabolised by them. The conditions of the ethanol-producing processes of the invention will typically be adapted and optimised for a suitable level of ethanol production. Such a level of ethanol production will typically be one that is as high as possible without killing or impairing a high proportion of the yeast cells. Ethanol will typically be produced by a fermentation broth that comprises yeast cells according to the invention, water, one or more sources of xylose and other nutrients. In particular, the presence of an appropriate concentration of glucose is desirable as it facilities the growth of the yeast cells. Also, the presence of Mn 2 ~ or Mg 2 * ions is desirable as one of these ions is necessary to the function of the barley xylose isomerase. Further, the presence of oxygen is desirable, especially in fermentation broths containing S. cerevisiae. The process may be carried at any temperature that facilities ethanol production but temperatures of from 15 to 40 "C are preferred and temperatures of from 30 to 35 'C are particularly preferred. For the ethanol-producing processes of the invention, it is preferred that transformed or transfected yeast cells, espe¬ cially those of S. cerevisiae and/or S. pombe are used, including the preferred strains mentioned above. In the ethanol-producing processes of the invention, the substrate is wholly or partly converted to ethanol, which may be recovered by any suitable means known in the art. The present invention also provides a process of producing a polypeptide having xylose isomerase activity. Such a process will typically comprise transforming or transfecting host cells with vectors comprising nucleic acid sequences according to the invention and expressing the nucleic acid sequence in these cells. In this case, the nucleic acid sequence will be operably linked to a promoter capable of directing its expres¬ sion in the host cell. Desirably, such a promoter will be a "strong" promoter capable of achieving high levels of expres¬ sion in the host cell. It may be desirable to overexpress the polypeptide according to the invention in the host cell. Suitable host cells for this purpose include bacterial cells, for example E. coli cells, and yeast cells, for example those of the preferred species and strains referred to above. The thus produced polypeptide of the invention may be recovered by any suitable method known in the art. Optionally, the thus recovered polypeptide may be purified by any suitable method, for example a method according to Sambrook et al. (Molecular Cloning: A Laboratory Manual). A further embodiment of the present invention is a process of producing xylulose by contacting a substrate containing xylose with a polypeptide of the invention having xylose isomerase or cells according to the invention that express such a polypep¬ tide. In such a process, the polypeptide according to the invention isomerises xylose to xylulose, which may be recov¬ ered by any conventional method. The nucleic acid sequences of the invention may be used to prepare probes and primers. These will be useful in the isolation of xylose isomerase genes having sequences similar to that of SEQ. ID. No. 2. Such probes and primers may be of any suitable length, desirably from 10 to 100, for example from 10 to 20, 20 to 50 or 50 to 100 bases in length. Two particularly preferred pri-mers are those shown in SEQ. ID. No. 4 and 5. The present invention also provides antibodies to the polypep¬ tides of the invention, specifically antibodies to the native xylose isomerase protein. These antibodies may be monoclonal or polyclonal. For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv, F(ab' ) and F(ab' ) 2 fragments, as well as single chain antibod¬ ies. The antibodies may be produced by any method known in the art, such as the methods of Sambrook et al. (Molecular Cloning: A Laboratory Manual; 1989). For example, they may be prepared by conventional hybridoma techniques or, in the case of modified antibodies or fragments, by recombinant DNA technolo ¬ gy, for example by the expression in a suitable host vector of a DNA construct encoding the modified antibody or fragment operably linked to a promoter. Suitable host cells include bacterial (for example E. coli), yeast, insect and mammalian cells. Polyclonal antibodies may also be prepared by conven¬ tional means which comprise inoculating a host animal, for example a rat or a rabbit, with a peptide of the invention and recovering immune serum. The following Examples illustrate the invention. EXAMPLES Example 1 Purification, sequencing and characterisation of the xylose isomerase protein. Materials & Methods Materials The starting material for the purification of xylose isomerase enzyme was three to four days old malted barley obtained from Lahden Polttimo (Lahti, Finland). All used column materials were Pharmacia Bioproducts. Sorbitol dehydrogenase and NADH were obtained from Boehringer Mannheim. Xylulose, TPCK (N-tosyl-L-phenylalanine chloromethyl ketone) -treated trypsin, polyvinylpyrrolidone (PVP), XAD-4 (an amberlite non-ionic polymeric adsorbent) and Triton X-100 were Sigma products. Barley seeds (Hordeum vulgare, cv himalaya) were used as a source for mRNA and genomic DNA isolation. Surface-sterilized seeds were first germinated for 3 to 4 days in the dark at 20 ' C on 0.7 % water-agar. Methods Xylose Isomerase Assay Xylose isomerase activity was determined by a two-step proce¬ dure where the xylulose formed during the first step was reduced in the second step to xylitol by sorbitol dehydrogena¬ se with a concomitant oxidation of NADH (Callens et al. (1982): Enzyme Microb. Technol. 8, 696-700). The standard assay mixture was at pH 7.2 and contained 10 mM Tris-HCl, 10 mM MnCl.- and 50 mM D-xylose in a 100 μl reaction volume. After one hour incubation at 35 ' C the isomerase reaction was stopped by adding 900 μl ice-cold 0.1 M triethanolamine buffer, pH 7.0. The isomerase reaction product, D-xylulose, was subsequently reduced at pH 7.0 to D-xylitol in a reaction vessel containing 33 μg/ml sorbitol dehydrogenase and 16.8 μM NADH. The amount of NADH oxidised was measured with an AKEA- analyzer, thus providing a measure of the amount of xylulose produced, which is proportional to enzyme activity. Protein Assay Protein concentrations were routinely estimated at 280 nm using adsorptivity coefficient of 0.96 mg- 1 cm- 1 . Electrophoretic procedures SDS polyacrylamide gel electrophoresis (PAGE) of protein fractions was carried out as described by Laemmli (1970: Nature 227, 680-685). Western analysis was performed accord¬ ing to the manufacturer' s instructions using a " Proto Blot" system supplied by Promega. Enzyme purification In order to choose the material for the enzyme purification, one to six days malted barley seeds were analysed by measuring their total protein content and xylose isomerase activity. The results are shown in Fig.1. Based on these results, three to four days malted barley seeds were chosen as a starting material for the purification. The enzyme has an obligatory requirement for Mn 2 * or Mg 2 *, one or both of which were included in all the buffers used. In order to improve the release of enzyme from the seeds, 0.1 % Triton X was used in the extraction buffer and PVP and XAD-4 were included to minimize detrimental reactions between the phenols and pro¬ teins. Protein purification steps were carried out at 4 " C. Chromatog¬ raphy on Superose 12, Mono Q and Phenylsuperose were performed with FPLC system (Pharmacia). Eluates were monitored at 280 nm. Step 1. Crude protein extract and ammonium sulphate fraction- ation 500 to 1000 g of three to four days old malted barley was ground slightly in a pH 7.0 buffer containing 10 mM Tris-HCl, 1 mM cysteine, 1 mM MnCla (Buffer A). After breaking the seeds 0.1 % (v/v) Triton X-100, 0.1 % (w/v) PVP and 0.1 % XAD-4 (w/v) were added and the mixture was blended continuous¬ ly for 60 to 75 minutes at +4 ' C. The extract was filtered through cheesecloth and centrifuged for 20 minutes (8000 rpm, GSA rotor, Sorvall centrifuge). The supernatant was subjected to ammonium sulphate fractionation. Protein precipitating between 33 % and 60 % saturation with ammonium sulphate was collected by centrifugation as above and dissolved in 100 to 200 ml of pH 7.0 buffer containing 10 mM Pipes, 1 mM cysteine, and 1 mM MnCl_> (Buffer B). Step 2. Sephadex G-25 gel filtration In order to remove phenols and other small molecules, the extract was filtered through a Sephadex G-25 column (90 cm x 5 cm) (Strobaek et al. (1976): Carlsberg Res. Commun. 41, 57-72) which was equilibrated with buffer B including 0.1 M NaCl. The flow rate was 10 ml/min and the protein fraction after the void volume was collected. Step 3. DEAE ion exchange chromatography A DEAE (Diethylaminoethyl ) Sephadex column (20 x 2.9 cm) was equilibrated with the buffer B including 0.1 M NaCl. The protein fraction from G-25 gel filtration was applied into the gel and the column was washed with the same buffer. The proteins were eluted with a linear gradient of 0.1 M NaCl to 0.25 M NaCl in buffer B (400 ml). The flow rate was 30 ml/h and fractions (4 ml) containing the xylose isomerase activity were pooled and concentrated by filtering through an Amicon P-30 filter. The concentrated material was dissolved in buffer A and applied into the affinity column. The xylose isomerase was eluted from DEAE ion exchange column with 0.18 M to 0.22 M NaCl, the peak activity eluting with 0.19 to 0.2 M NaCl. Step 4. Affinity chromatography The affinity column (2.0 x 10 cm) was prepared by adding xylose sugar into epoxy-activated Sepharose 6B. The addition was performed according to the manufacturer' s instructions. The column was further equilibrated with buffer A. After absorbing the pooled and concentrated crude material from the previous step, the column was washed with buffer A and the enzyme was eluted using the same buffer with 1 M NaCl. Frac¬ tions containing xylose isomerase activity were concentrated using a Centricon 30 filtering unit according to the manufac¬ turer' s instructions. The affinity column required the pre¬ sence of Mn 2 - and worked best in buffer A. Step 5. Gel filtration on Superose 12 A Superose 12 column was equilibrated with a pH 7.0 buffer containing 20 mM Pipes, 1 mM cysteine, 1 mM MnCla (buffer C) including 50 mM NaCl at a flow rate of 25 ml/h. Aliquots containing 5 to 10 mg of protein in 200 μl of running buffer were applied into the column and the elution was performed with the same flow rate. The fractions containing xylose isomerase activity (0.58 ml) were pooled and concentrated with Centricon 30 microconcentrators (Amicon). In the Superose 12 gel filtration, the xylose isomerase activity was eluted from the column in the volume 12.18-13.34 ml (fraction 21-23). The recovery varied from 84 % to 89 % in the conditions used. Step 6. Ion exchange chromatography on Mono Q A Mono Q column was equilibrated with buffer C at a flow rate of 1 ml/min. The pooled and concentrated fraction (150 μl ) from Superose 12 chromatography was applied into the column and washed with 5 ml of buffer C. The activity was eluted with a linear gradient (20 ml) of 0 to 0.3 M NaCl in buffer C. The pooled Mono Q fraction was run on a 10 % SDS PAGE and stained with Coomassie Brilliant Blue. The fractions (250 μl ) containing the isomerase activity were pooled and further used for protein sequencing and antibody production. The thus purified material was about 90 % homogeneous. The results of the purification procedure are given in Table 1. Table 1. Purification of xylose isomerase Volume Total Total Specific Purific. (Ml) Activity Protein Activity Factor μmol/h mg μmol/mg Extract 1000 704 17600 0.04 1 Step 1 140 910 3556 0.26 6. 5 (60% AmS) Step 2 320 448 1964 0.23 5. 75 (G-25) Step 3 81 356 103 3.48 87 (DEAE) Step 4 7 210 3.85 54.5 1364 (Affinity) Step 5 3. 5 178 1.45 122.7 3050 (Superose 12) Step 6 1. 25 146 0.1 1462.0 365. 62 (MonoQ) Peptide Digestions, Purification and Sequencing The xylose isomerase enzyme isolated as above was further purified for the peptide digest using Bakerbond C-18 reverse phase column in the HPLC system. Protein was bound in 20 % acetonitrile (ACN)/0.1 % trifluoroacetic acid (TFA) -water solution. The flow rate was 0.6 ml/min and the peak absorbing at 218 nm (16.91 min) was collected and dried in a vacuum using a Speed Vac-system. This sample was further used in peptide digestions with TPCK-treated trypsin. The digest took place in 1 % ammoniumbicarbonate with 2 % (w/w) trypsin for two hours at 37 * C after which more trypsin 2 % (w/w) was added and the reaction was continued overnight. The peptides were purified using Bakerbond C-18 reverse phase column and the linear gradient of 0-60 % ACN/0.1 % TFA was run at flow rate of 0.60 ml/min. The peaks absorbing at 214 nm were collected manually and applied to the Beckman 890D amino acid sequencer. The following ten peptide sequences were obtained and all were present in the polypeptide sequence (SEQ. ID. NO. 3) of the enzyme deduced from the xylose isome¬ rase cDNA (SEQ. ID. NO. 2) as shown in figures 9A and 9B. Peptides 431, 439 and 443 each had one amino-acid mismatch deduced from the cDNA sequence. 435 I Phe-Gly-Leu-Thr-Gly-Glu-Phe-Lys (SEQ. ID. No. 9) II Glu-Gly-Tyr-Gln-Thr-Leu-Leu-Asn-Thr-Asp-Met-Lys (SEQ. ID. No. 10) These two peptides were mixed in a single sample but because they were present in widely differing amounts both sequences could be deduced unambiguously. 434 Tyr-Met-His-Gly-Ala-Ala-Thr-Ser-Pro-Glu-Val-Lys (SEQ. ID. No. 11) 433 (Ser or Trp)-Tyr-Asn-Ala-Glu-Glu-Val-Ile-Leu- (Val ) -Gly-Lys (SEQ. ID. No. 12 and SEQ. ID. No. 13, respectively) 432 Ala-His-Phe-Glu-Phe-Met-Glu-Lys (SEQ. ID. No. 14) 431 Gly-Thr-Gly-Gly-Val-Pro-Phe-Gly-Ala-Pro-Thr-Lys (SEQ. ID. No. 15) 443 Xxa-Xxb-Xxc-Xxd-Glu-Leu-Glu-Thr-Ala-Arg (SEQ. ID. No. 16) Xxa means Met or Ser, Xxb means Tyr or Pro, Xxc means Ala or His and Xxd means Tyr or His. 439 Met-Lys-Asp-Xxx-Leu-Arg (SEQ. ID. No. 17) (Xxx stands for "any amino acid" ) 440 Asn-Asp-Gly-Leu-Ala-Pro-Gly-Gly-Phe (SEQ. ID. No. 18) 444 Ile-Asn-Tyr-Glu-Gly-Pro-Thr-Ser-Lys (SEQ. ID. No. 19) Further characterisation of the enzyme Effect of temperature on enzyme activity Xylose isomerase activity was measured on the temperature range 25-100 'C (Fig.3). The results from the assays made above 60 * C were corrected for the non-enzymatic isomerization that occurs at such temperatures. Fig. 2 shows the effect of the temperature on the xylose isomerase activity. The tempera¬ ture optimum is 60 * C and above 80 * C the enzyme lost its activity. The enzyme was quite stable and preserved its activity after a 5 hour incubation at 60 " C. Effect of pH on enzyme activity The effect of pH on the activity of the enzyme was measured in the pH range 4 to 13 (Fig. 3). The following buffers were used 0.05 M acetate/NaOH (pH 4 to 5.6). 0.02 M Bis-Tris (pH 5.5 to 7.0), 0.02M Pipes (pH 6.5 to 7.5). 0.05 M Triethanola- mine/NaOH (pH 7.0 to 8.5), 0.010 M Tris-HCl (pH 7.0 to 9.0), 0.05 M glycine-NaOH (pH 8.5 to 10.0), 1 M sodium carbonate/bi¬ carbonate (pH 9.2 to 10.5), 0.025 M disodiumphosphate/NaOH (pH 11 to 12), 0.05 M NaOH/KCl (pH 11 to 13). Above pH 9.5, corrections were made for the non-enzymatic isomerization of D-xylose that occurs under such conditions. The enzyme preserved its activity in the pH range 5.5 to 10.5. Its activity was high across the pH range of 7.0 to 9.0. Molecular weight The molecular weight of the enzyme was determined as 100 000 dalton by its elution volume from a calibrated Superose 12 column. SDS/PAGE of the purified xylose isomerase gave a molecular weight estimation of 50 000 suggesting that the native protein is a dimer with two subunits. Molecular weight calculations based on the cDNA sequence gave the estimate 53 620. Enzyme Specificity The specificity of the enzyme for xylose, as opposed to glucose and ribose, was determined by measuring its activity when presented with each of the three sugars. No glucose isomerase or ribose isomerase activity was observed, which suggests that the enzyme has a high specificity for xylose. Enzyme activity was measured in the following manner in each case. A two-step procedure was employed in which the sugar was reduced by sorbitol dehydrogenase with concommitant oxidation of NADH (Callens et al. (1982): Enzyme Microb Tech- nol. 8, 696-700) The assay mixture was at pH 7.2 and contained 10 mM Tris-HCl, 10 mM MnCl 2 and 50 mM of the relevant sugar in a 100 μl reaction volume. After a one hour incubation at 35 ' C, the reaction was stopped by adding 900 μl ice-cold 0.1 M triethanolamine buffer at pH 7.0. The reaction product was subsequently reduced at pH 7.0 in a reaction vessel containing 33 μM NADH. The amount of NADH oxidised was measured with an AKEA analyser, in order to give a measure of enzyme activity. Example 2 Antibody Production The purified xylose isomerase protein (Mono Q fraction) was used as an antigen and a rabbit was immunized with 20 μg of protein. The immunization was repeated after two and six weeks with the same amount of protein. Blood samples were collected 7 to 10 days after the injection and the final blood sample was collected 7 to 10 days after the last antigen injection. Example 3 Isolation of cDNA and gene coding for xylose isomerase DNA manipulations Restriction endonuclease digests, Southern blot analysis and other techniques were performed according to standard proce¬ dures (see, for example, Sambrook et al. : Molecular Cloning: A Laboratory Manual; 1989). Isolation of Poly(A) RNA The scutella from two to three days old germinated barley seeds were separated and powdered in liquid nitrogen in a mortar. pH 9.0 buffer containing 0.05 M Tris-HCl, 0.01 M EDTA, 0.1 M NaCl, 2 % (w/v) SDS, and 2 mg/ml proteinase K was added into the powder (10 ml per g of scutellum). The solution was further homogenized with a Polytron homogenizer and incubated for 30 minutes at 40 to 50 " C. After the incubation, the solution was extracted four to five times with the same volume of a solution containing (1: 1 phenol and chloroform) : isoamy- lalcohol (24: 1) and finally with chloroform : isoamylalcohol (24: 1). Between the extractions the water phase was separated by centrifugation for 15 minutes (4000 rpm, GSA rotor, Sorvall centrifuge). One tenth volume of 4 M NaCl solution and 2 ml of oligo dT-cellulose was added into the protein-free water phase, mixed for 15 minutes and centrifuged in a table top centrifuge at 3000 rpm. The cellulose was washed three times with a pH 7.9 buffer containing 0.01 M Tris-HCl, 0, 4 M NaCl, and 0.2 % SDS. After the washings the cellulose was poured into a column equilibrated with a pH 7.5 buffer containing 0.1 M Tris-HCl, and 0.1 M NaCl. The column was eluted with prewar ed 0.1 M Tris-HCl, pH 7.5 buffer and five 3 ml frac¬ tions were collected. Poly(A) -RNA was precipitated by adding one tenth volume of 3 M sodium acetate solution and 2 volumes of cold ethanol and keeping the samples at -20 "C overnight. Preparation and screening of cDNA library 5 μg of the poly(A) RNA as a template cDNA was synthesized using a Promega Kit (Promega, WI, USA) using the components of cDNA synthesis kit supplied by Boehringer Mannheim and follow¬ ing the supplier' s instructions. The double-stranded cDNA was treated with T4 polymerase and ligated with EcoRI-adapters and this was further ligated to the vector. Several libraries were prepared using different vectors including gtll, gt 10 and ZAP vectors which were all packed using a "Gigapack Gold II" packing kit supplied by Stratagene. The screening of the libraries were performed using the oligomers synthesised against the peptide sequences as a probe. The positive phage clones picked by oligomers from the gtll library were further confirmed using antibody screening. After growing for three hours the phages were transferred onto a nitrocellulose filter and hybridized with the antibody using a "Proto Blot" system supplied by Promega. Isolation of Full-length cDNA by means of PCR (Polymerase chain reaction) In order to get a full-length cDNA the RACE protocol designed for PCR (Frohman in "PCR Protocols": eds Innis et al. , Aca¬ demic Press, 1990) was used. The first strand of cDNA was syn- thesised using the anchor (dT)-oligomer (5' -TTACTCGAGAATTCATC- GA(dT)ιτ -3' (SEQ. ID. No. 4) as a primer instead of pure oligo(dT). cDNA thus obtained was used further as a template in a PCR reaction and the anchor sequence and the known sequence corresponding the nucleotides 620-644 (5' -TTATGGGGAACTGCACAACTTTC-3' ) (SEQ. ID. No. 5) within the cDNA sequence were used as primers. The reaction mixture and the amplification conditions were as described by Frohman (1990). The fragment synthesized in the PCR reaction was purified by agarose gel electrophoresis and cloned using a "TA" cloning kit supplied by Invitrogen. Three clones were analyzed by sequencing. Isolation of total genomic DNA Total genomic DNA was isolated from the shoots of four to five days old malted barley according to the method described by Dellaporta et al. (1983: Plant Mol. Biol. Rep. 1 (4), 19-21). The isolated total DNA was purified on CsCl gradients with ethidium bromide (10 mg/ml). After removing the ethidium bromide by isoamylalcohol extraction the DNA was dialysed against a 0.010 M Tris-HCl, pH 7.5, 0.001 M EDTA buffer. Isolation of xylose isomerase gene by PCR Based on Southern hybridizations with cDNA it was concluded that the isomerase gene was several kilobases in length. Therefore, the gene was isolated in two smaller fragments and the cDNA sequence was used as a basis for the primer sequences in PCR reactions. Using the primers corresponding to nucle¬ otides 5-30 (sense) and 620-645 (antisense) of the cDNA about two kilobase DNA fragment was obtained which was further cloned using Invitrogen " TA" cloning kit. The genomic area corresponding the 3' -end of the gene was obtained using the primers corresponding to nucleotides 620-645 (sense) and 25 nucleotides preceding the poly(A) tail (antisense) in the cDNA. The fragment obtained was 2.5 kilobases in length and was cloned as above. Three clones for each 5' or 3' fragment were sequenced. DNA sequencing Positive cDNA clones were sequenced using sequencing kits supplied by USB. Sequencing reactions were analyzed in polyacrylamide gel electrophoresis using an LKB electrophore- sis system. PCR clones were sequenced using fluorescently labelled primers and sequencing kits supplied by Applied Biosystems. Reactions were analyzed using Applied Biosystems 373 A automatic sequencer. Oligonucleotide synthesis The oligonucleotide sequences corresponding to the peptide sequences were synthesized using Applied Biosystems DNA synthesizer 381A. Isolation and structure of cDNA clones encoding xylose isome¬ rase Xylose isomerase cDNA clones from the gtll library were identified by hybridization with the oligomers based on the peptide sequences 435 I, 435 II, 434, 431 and 432 as probes. The positive clones were further confirmed by the antibody screening. The sequence of a cDNA clone with an insert of 990 bp is depicted in SEQ. ID. No. 2. This sequence contained the 5' end of xylose isomerase cDNA up to the EcoRl restriction site. In this area all except three (435 I, 443 and 440) peptide sequences were found. The library was screened with the oligomers for these missing sequences but without any positive result. In order to obtain the missing part of the cDNA a RACE PCR was performed. Two of the three PCR clones obtained contained the area from the 5' primer starting point up to the end including the poly A tail. Clones had an insert of 750 bp and this insert contained also the missing three peptide sequences. The peptide sequence 435 I crosses the end of the previously isolated fragment and the beginning of this new fragment. Assuming that translation starts with methionine, there is a 75 nucleotide non-translated area at the 5' end of the full- length cDNA. The non-translated area at the 3' -end of the cDNA is 192 nucleotides and contains three pol (A) addition signal sequences and several stop codons. The full-length cDNA is 1710 nucleotides in size which corresponds with the results obtained with Northern hybridization when the first isolated fragment was used as a probe. The 480 amino acids encoded by the cDNA have a molecular weight of 53 620, which is in good agreement with the values obtained for the protein. Genomic DNA coding for xylose isomerase Three PCR clones, each containing 5' or 3' ends of the gene were sequenced. A genomic fragment of 4473 bp in length corresponding to the xylose isomerase cDNA sequence was revealed. The genomic sequence was interrupted with 20 intron sequences (Fig. 4). In most cases the exon sequences are quite short compared to the intron sequences. The 5' non- translated area is for example interrupted with a long intron of 400 nucleotides. In SEQ. ID. No. 1 the whole nucleotide sequence of the xylose isomerase gene is shown. In Figure 5 all 20 exon-intron junctions are represented which shows that every intron starts with GT and ends with AG, thus following the universal exon-intron rule. Example 4 Transformation of yeasts by the isomerase cDNA Materials and methods Microbial strains and plasmids Escherichia coli strain XLl-Blue (Bullock (1987): Biotech- niques 5, 376-378) Stratagene, La Jolla, CA, USA) was used for propagation of plasmids. The cloning vector was pCRTMII (Invitrogen, San Diego, CA, USA). Plasmids for transforma¬ tions were constructed by using pAAH5 (Ammerer (1983): Meth Enzymol. 101, 192-201) for Saccharomyces cerevisiae and pEVPll (Hildebrandt et al. (1989): FEBS 243(2), 137-140) for Schizosac¬ charomyces pombe as basic vectors using standard recombinant DNA techniques. Saccharomyces cerevisiae strain YF135 (deposit number CBS 601.94) and Schizosaccharomyces pombe strain ALKO 2185 (deposit number CBS 602.94 were used as recipients for transformations. The plasmid pALK252 is constructed from pAAH5 by removing the restriction site Xhol. Growth media and culture conditions E. coli strains were grown in L-broth (Sambrook et al. (1989): Molecular Cloning: A Laboratory Manual) supplemented with 50 μg/ml ampicillin when needed. Cultures were grown up at 37 * C overnight. YPD agar slants (1 % yeast extract, 2 % peptone, both from Difco and 2 % glucose from Merck) were used for storing the Saccharomyces and Schizosaccharomyces strains. The plates and media for Saccharomyces and Schizosaccharomyces transformations with leucine selection were 2 % glucose (Mer- ck)/0.67 % yeast nitrogen base without amino acids (from Difco) + amino acids lacking leucine (YNBLeu-). DNA techniques DNA manipulations were performed by standard techniques (Sambrook et al. (1989): Molecular Cloning: A Laboratory Manual). The restriction enzymes T4 DNA ligase and Klenow fragment of DNA polymerase I were from Boehringer (Mannheim, Germany) and New England Biolabs, MA, USA). Each enzyme was used according to the supplier' s recommendation. Plasmid DNA from E. coli was isolated by using Qiagen columns (Diagen GmbH, Germany) or the Magic Minipreps DNA Purification System (Promega, Madison, WI, USA) according to the supplier' s instructions. DNA fragments for transformations were isolated from low melting point agarose gels (FMC Bioproducts, Rockland, ME, USA) using beta-agarase from New England Biolabs (Beverly, MA, USA). Transformation of E. coli strain XLl-Blue was performed by the supplier's suggested method (Stratagene, La Jolla, USA). For Southern blot analysis the DNA was transferred from agarose gels to nylon membranes by VacuGene TM XL apparatus (Pharmacia, Uppsala, Sweden). The labelling of the probes with digoxigenin and hybridization of the filters were done according to , he procedures of Boehringer (Mannheim, Germany). The PCR reactions were performed by Programmable Thermal Controller PTC-100TM (MJ Research Inc. , Watertown, Massachu¬ setts, USA) in 100 μl volumes. The reaction mixture contained 0.1 mM of each dNTP (Cetus), 5 ng of each primer and 50 ng of plasmid template in 1 x buffer supplied by Dynazyme. The protocol used was the following: incubation a 95 ' C for 5 min before adding the F-500L Dynazyme (1 unit, Finnzymes, Espoo, Finland) and 100 μl of paraffin oil; denaturation at 95 * C for 1 min, annealing at 50 ' C for 1 minute; extension at 72 ' C for 2 min, with a total of 30 cycles each comprising all of these steps. The PCR fragments were purified using the "Magic PCR Preps" DNA Purification System (Promega, Madison, WI, USA). The oligonucleotides used were sythesized using an Applied Biosystems (Foster City, USA) 381A Synthesizer. Sequencing was performed directly from the plasmids using an Applied Biosystems Model 373A automatic DNA sequencer. Transformations of Saccharomyces cerevisiae and Schizosaccharo¬ myces pombe were carried out by the method of Elble (1992: BioTechniques 13(1) 18-20). Example 5 Construction of Saccharomyces cerevisiae and Schizosaccharomy¬ ces pombe strains carrying the cDNA coding for the xylose isomerase gene. The cDNA encoding the barley xylose isomerase was cloned in two fragments. A lkb gtll clone was found to encode the amino terminal part of the barley xylose isomerase. The 1 kb EcoRI -insert was initially transferred into the EcoRI site of pBR322 (pALK705). A unique site for the restriction enzyme StuI was found within the 5' untranslated region. lkb Stul- EcoRI fragment was isolated from pALK705 and ligated to Smal-EcoRI-digested Bluescript SK+ (pALK709) (See Figure 6). The cDNA clone encoding the carboxy-terminal portion of the barley xylose isomerase was cloned by using specific primers and RT-PCR and a 0.7 kb EcoRI fragment was inserted into EcoRI-digested Bluescript SK+ (pALK706). To obtain a full length cDNA the carboxyterminal portion of cDNA from plasmid pAlk706 was isolated and subsequently ligated to the EcoRI site of the plasmid pALK709 linearized with EcoRI to generate plasmid pALK710. The correct orienta¬ tion of the insert was confirmed with DNA sequencing. The full length cDNA was transferred into a Saccharomyces cerevisiae expression vector under the control of the alcohol dehydrogenase promoter using PCR. A forward primer containing a BamHI restriction site and covering the initiator ATG codon and seven additional codons 5' AAAAGGATCCATG AAG GGC GGG GAG CTC CTG GTC 3' (oligo 51: SEQ ID. No. 6) and a reverse primer (oligo 50: SEQ ID No. 7) 3' C TAC GAC AAG GTT AGG CGA GAC ATC CCTAGGAATA 5' covering the 3' end of the coding region and containing a BamHI restriction site were synthesized and used in the PCR with pALK710 as the template. The PCR product was digested with BamHI and the ends were filled with dNTPs using the Klenow fragment of the DNA polymerase I. The 1.5 kb fragment was isolated and ligated into HindiII digested and blunt-ended pAAH5 to generate plasmid pALK721 (See Figure 7). The expression vector pEVPll containing the Schizosaccharomy¬ ces pombe adhl promoter was linearized with BamHI and ligated with the 1.5 kb BamHI digested PCR product, resulting the expression construct pALK720 (See Figure 7). After propagation in E. coli, the vectors were transformed into yeast cells using the simplified Li-method of Elble (1992: BioTechniques 13(1), 18-20). The transformants were selected by complementation of the leucine auxotrophy of the host strain. Example 6 Construction of Saccharomyces cerevisiae and Schizosaccharomy¬ ces pombe strains carrying the xylose isomerase gene and the Hormoconis resinae glucoamylase signal sequence. Construction the plasmids pALK724 and pALK725 (Fig 8). A new PCR primer with Nael restriction site (5' ATTAAGCC GGC GGG GAG CTC CTG GTC 3' ) was synthesized and called oligo 54 (SEQ. ID. No. 8). A PCR reaction was conducted using oligos 54 (SEQ. ID. No. 8) and 50 (SEQ. ID. No. 7) using pALK710 as a template. The PCR product was digested with Nael and BamHI. A 1.5 kb fragment was isolated and ligated into StuI-BamHI digested pALK730 to generate pALK723. pALK730 is a Bluescript SK+, containing the Hormoconis resinae glucoamylase 5' untranslated region signal sequence and the prosequence in a 120 bp fragment. This signal sequence is capable of directing secretion of heterolo- gous proteins, such as the barley xylose isomerase from yeast cells. The blunt ended insert can be joined in frame after Lys-Arg codons. The 1.5 kb insert was separated from pALK723 with restriction enzymes Clal and BamHI, ends were filled with dNTPs and the blunt- ended fragment was ligated into HindiII digested and blunt-ended pAAH5 to generate the expression construct for S. cerevisisae and to BamHI digested and blunt ended pEVPll to generate the expression construct for S. pombe. Example 7 Detection of the xylose isomerase gene in transformed yeasts 5 μg of yeast cells from a YNBLeu- 2 % Glc plate were suspend¬ ed into 80 μl cell-lysis buffer: 50 mM Tris-HCl pH 8.0/ 2.5 M LiCl/4 % (v/v) Triton X-100.62.5 mM EDTA and 40 μl of phenol, 40 μl of chloroform and 0.1 g washed glass beads were added. After vortexing the suspension for 1.5 min, the broken cells were centrifuged at 15 000 rpm for 2 min. To the supernatant 80 μl of isopropanol was added. The mixtures were kept 10 min at -20 * C, and centrifuged of 13 000 rpm for 10 min at 4 ' C. The precipitates were washed with 70 % (v/v) ethanol, and the remaining ethanol was removed in Speed-vac evaporator. 20 μl 10 mM tris HCl/0. ImM EDTA buffer was added. 1 μl of the suspensions were used in PCR reactions as described above. The primers were the oligos 50 and 51 described above. 10 μl of the PCR products from S. cerevisiae transformed with pALK 720, from S. pombe transformed with pALK 720, from S. cerevisiae transformed with pALK721 and from S. cerevisiae transformed with pALK724 were run on an agarose gel, and stained with ethidium bromide. The resulting bands showed that the xylose isomerase gene was present in the transformed yeast cells. The standards used in this gel were molecular weight marker III from Boehringer Mannheim. The PCR product identified was about 1.4 kb in length which corresponds to the length expected from the sequencing of the cDNA. The length of the full-length cDNA is 1710 base pairs (see above) and the 1.4 kb fragment corresponds to a 1473 base pair length of cDNA lacking the 3' untranslated region and promoter. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Primalco Ltd. (B) STREET: Valta-akseli (C) CITY: Rajamaki (E) COUNTRY: Finland (F) POST CODE (ZIP) : 05200 (ii) TITLE OF INVENTION: Xylose Isomerase (iii) NUMBER OF SEQUENCES: 19 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4473 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Hordeum vulaqare cv himalaya (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TCCGCTTAAC CAACCCCGCA CGGGTCGCGG ACCTGGTTTC CTCCTCCGGC GGCCGCCGCG 60 GTGAGCCCCT CCGCCTCCTT CCTCCCATGC TTCTTCCTCC TCCTCCGCAG ACCCCCTCAG 120 TAGCGGGGAT CCCCCGTTGG CGGCAACCAT CCGTCGGCGC CGTCGTGTCG TCGCTTCGCC 180 GTGACAGAAC CCCATCTCAA GTCTTCTTCG TCGCCAACCC GTCCAGTTTG TGGCCGCGGG 240 CACCATGCCT TAGCTTCATG CCGGAGTACC AGTAGTCATA CAAGCTGTTT ACTTTGCAAA 300 AAATAAATTG GGGATCCGTT CCAATCGAGG GCCCGTTCTT GCTTAGGACA GGCTTGGTAG 360 TTAGGAC CG CGGAATTGGA AAGGTTTCGT TCCGGAGATT TGGATCCTCG TACGGAACAC 420 TTAACTTAAC TTTGTCGATT ATAATTTCTC TTGTTTGAAG TTGCAGAGGC CTTGCGCCAT 480 GAAGGGCGGG GAGCTCCTGG TCCTGCTGCT GGCCTCGTCC CTCTGCCTGT CCGCCGCGGT 540 GAGCGATTGC CAGCTTTGCG GCCTTCTTTG CGTGATTGAG ATGCCCATGG TGGTTTGATC 600 TATCAACTTG ATGCGATCCT GGCTCTGATC TGGCTGCTCC TCGCAGGTTG CCGCGCAGGA 660 AACCTGCCCG GCCGACATCG GCGCCAAGTG CACCGATGCC GCCTCCGATG ATTGGGAGGG 720 CGAGTTCTTC CCCGGCATTG ACAAGATCAA CTATGAGGTG AGGCCATTGA CCTTGGACTG 780 GTGCTAAACA GATTATCTTC ACGGTTCGCT TTCAGTTTAA TAGCTTTTGT CCTTGAACAA 840 TTTCGCCGCA GGGTCCTACC AGCAAGAAGC CGCTTTCTTA CAAGTGGTAT AACGCGGAGG 900 AAGTGATCCT CGGAAAGAAA ATGAAGGTAT GGTTTGCTTT GCAGAACCAT GTCTCGTTGG 960 TGGTAACATC ATTTTAATTT GCTTTTTTAC ATCATGTGCT ACAGTAGGAT TATATATTCG 1020 TTTTTGTGCT ATAGGATTGG TTTCGGTTCA GCGTGGCGTT TTGGCATACG TTCCGGGGTA 1080 CTGGAGGAGA TCCCTTTGGT GCACCTACGA AGAACTGGCC TTGGGAGGAT GGCACCAATT 1140 CCTTGGCCAT GGCTAAGAGA AGAAGTATGA GGATTACTTT ATCTTGTTCT TTTAGATCCA 1200 TGGCGTTATG AAATCGCATA TTTTCTGGAA TCAACAACTT TTATGTTTGG ATTTCTGTTT 1260 CAGTGAAAGC TCACTTCGAG TTCATGGAGA AGCTTGGAGT TGAAAGGTGG TGCTTCCATG 1320 ACAGGGACAT CGCCCCTGAT GGCAAAACAC TCGCGGTATG ATTTTTTCAG AATGATTGCT 1380 GAAGTTGCAA GTTGCAAACC TGAGTTAGGG AGTTGGAGAC TGGTAGTCAC CGATATTAGA 1440 GCAGTTTACT TATGATTATT TACAGGAGTA CAGGACTATG TTTTCAGCAC AAGGGATATG 1500 GTTGCTGATG TTGTACGGCT AATTTTATAC TTGCCATGTA ACTAAAAAGA ATACTCCCTC 1560 CGATCCATAT TAATTGTCGC TGATTTACTA AATCAGCGAC AATTAATATG ACTCGGAGGG 1620 AGTACTTGTT TTGGATCATC TATTCAAGGG CACAATAGAA CCAAACTGAT CACAATAATT 1680 TACAAGCAAT AACAAATATA AATGAACTTT ATTCCAGAAG ATTGCTTCTG TCTAAAATAG 1740 CATCTCTGGG TGTCACTGTA GTATATCTGA AAAATGCGTC GCCAAAATAT ATCTCAAGTG 1800 TGACTGTAAT TTGACTATCT TGGCTCCTAG GCCCTGAAAG GTCAGTATTT CTCCGTCTTA 1860 AGGGTCTTCT GTTCATATTG CAGGAAACAA ATGCTAACTT GGATGAGATA GTTGAGCTGG 1920 CAAAGCAACT CCAGGTTACT GCTACTACCT TAAAATGTTT CAACATTCAG GTTTCCTACA 1980 GTACAACTTT TTTCCGCTCT TTGCTCAAAA ATCTACTCTG AACATCATGT AGAGTGAGAC 2040 CAATATAAAG CCATTATGGG GAACTGCACA ACTTTTCATG CATCCACGTT ACATGCACGG 2100 AGCTGCTACT AGGTGGGTAC TAAATTTCAC TCCATTTGTG AATTTGAGTT ATGACTTGAA 2160 ACTAAGCAAG GCCTTCACTG ATGCTGCAGC CCAGAGGTCA AGGTGTATGC TTATGCTGCT 2220 GCTCAAGTGA AGAAAGCTTT GGAGGTGGGT TGTGTCTGAA TATCATAGGA AGCCTTCCTT 2280 TTGGTTAGTT GTTTAACTTG ATTTGTATCC CCTTCCAGGT TACTCACTAC CTAGGCGGTG 2340 AGAACTACGT ATTCTGGGGT GGAAGAGAGG GTTACCAAAC TCTTCTCAAT ACCGATATGA 2400 AGAGGGAACT TGAACATTTG GTATGGGTTG ACATACTTCT TGACTTTTGT TTGTGTTTCT 2460 ACTTGGTGTA GTTTGTAGCA CAAAAATTCT AGATTGGTAA CTTGTTCTTC TGTTGTGCAG 2520 GCTAACTTTC TTCAAGCTGC TGTTAACCAC AAGAAGAAGA TCGGCTTTAA CGGTAATTGC 2580 TTTTGCAGTG CGATAATTTG ATGTTCCTGG GTTCGAGGCT CCCAAATTTT ACAGTAAATA 2640 GGTATCATTG GTCCGCTCTA TGATAACCTT AAGGATACCT GCGGTGCCTG AATCCCCTTT 2700 TTAATTTCAG GAACATTGTT GATAGAGCCT AAGCCACAAG AACCAACAAA GCATCAGTAC 2760 GTTTGTCGTC TGGAAATATA TATTATGGAA CTGTGAACAG GACTGGAAAG AATTATACTT 2820 ATTTTTCTCT TGCAGGTATG ACTGGGATGT TGCAACTACA TTCTCTTTCC TACAGAAGTT 2880 TGGTCTTACA GGTATTTTTT GAAATGTGGC AAGAAAATCA TTTAGTACAA CCCTTCCTGT 2940 TTTTGTATGT TATGTGTGTA TTTGCAACAA TTCTATGCAA AATGAATCAT GTGAATTAAA 3000 CCATGTTTTC CTGACTATCA ACTTAGAACG TTCTATTTTG AAATAAAATG TATTCTTTTT 3060 TCTAATAATA CGGTGATGCA GGGGAATTCA AGATAAATGT TGAGTGCAAC CATGCTACTC 3120 TCTCTGGACA TAGGTCAGTT TCTTGCTCGC CTAAATCCAA TCATAACGGC TTAAGGTGAT 3180 ACTAGTACCA TAACTGCATC TAAAACTTGT GTCGTTCAGC TGCCATCACG AGCTTGAGAC 3240 TGCACGCATT AATGACATTC TTGGAAACAT TGATGCAAAC ACTGGTGATC CACAGGTTGG 3300 TATGTGTATA GTTCATAAAA TGGCTGTGAC TTGTTAAAAT CTCGGTTACA CGCTTAGTAG 3360 CTTAACATTT CAACTCTTAC GTGTCGGCAG GTTGGGACAC GGATGAGTTC CTTACAGACA 3420 TTTCAGAAGC TACCTTGATT ATGTCAAGTG TAGTTAAGAA TGTGAGTGGA ATTAATCATT 3480 TCTTGTACCT TTTCTGAACC AGAAGTCTAT TGTACTGTAT AATGTTTGTT TACCATTTTT 3540 ATTCAGGTAA CAGTACTCAT AATTTGTTCT GTTTGGTACG ATTGCAGGAT GGACTTGCGC 3600 CTGGTGGCTT CAACTTTTAC GCCAAATTGT ATGTTCCCAA ATAGCATTGG TGTTCCATGA 3660 ATTGAGCTTT GGTTCGACAA ATTTTTTGGC TGACATGGAA TTGTTTTGTG GTAACAGGCG 3720 GAGGGAGAGT ACTGATGTTG AGGACCTGTT TATTGCCCAT ATCTCTGGGA TGGACACCAT 3780 GGCCCGCGGC CGCCGCAATG TTGTCAAGCT GATTGAGGTA ACTGGAGACA TCATTAGTTC 3840 ATCACTCGGT AAAATTTGCA CATGCCTTTA CCTAAATGTA AGGTTTGTTT TCTATGTTAT 3900 TAGGATGGTT CCCTGGACGA GCTTGTACGC AAACGCTACC AGAGCTTTGA CACTGAGATT 3960 GGTGCCATGA TCGAGGTACA CAAATTAACA AGTTCGAATT TATTTTCTCA GAAGGATTGA 4020 ATTGGGATTG TGATACTGGA TTATGGTTTC GAACAGGCTG GGAAGGGCGA CTTTGAAACG 4080 CTAGAAAAGA AGGCCTTGGA GTGGGGCGAG CCAACCGTTC CATCGGGCAA ACAGGTAAAC 4140 GGAACGATAA ACCTATGGCA GCTCGTCTTT CACAGCACAA TGCAAATATA TTTCTGCGCC 4200 GATGATTGTT CCTTACGACC TTTTGTTGCC CGCTTCCCTA TGCAGGAATT GGCTGAGATG 4260 CTGTTCCAAT CCGCTCTGTA GATGGCGGCC CACGGTTCTA GGAATAAAAA AGCAAGAGCG 4320 CGACCTTGGA ACGCCCAGCC GTCCTCGTCA CTACAGGCGA TGTTCTATAG TTAGGCCTCC 4380 ATGCAGTGAA CCCTGTAAAC AAACTGCGTG GAGCTGAAAA TAATGTAACC TTATATCAAA 4440 ATTAAACTCG TTCTTCAACA CGGAATTTGG CTT 4473 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGHTH: 1710 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TCCGCTTAAC CAACCCCGCA CGGGTCGCGG ACCTGGTTTC CTCCTCCGGC GGCCGCCGCG 60 TTGCAGAGGC CTTGCGCCAT GAAGGGCGGG GAGCTCCTGG TCCTGCTGCT GGCCTCGTCC 120 CTCTGCCTGT CCGCCGCGGT TGCCGCGCAG GAAACCTGCC CGGCCGACAT CGGCGCCAAG 180 TGCACCGATG CCGCCTCCGA TGATTGGGAG GGCGAGTTCT TCCCCGGCAT TGACAAGATC 240 AACTATGAGG GTCCTACCAG CAAGAAGCCG CTTTCTTACA AGTGGTATAA CGCGGAGGAA 300 GTGATCCTCG GAAAGAAAAT GAAGGATTGG TTTCGGTTCA GCGTGGCGTT TTGGCATACG 360 TTCCGGGGTA CTGGAGGAGA TCCCTTTGGT GCACCTACGA AGAACTGGCC TTGGGAGGAT 420 GGCACCAATT CCTTGGCCAT GGCTAAGAGA AGAATGAAAG CTCACTTCGA GTTCATGGAG 480 AAGCTTGGAG TTGAAAGGTG GTGCTTCCAT GACAGGGACA TCGCCCCTGA TGGCAAAACA 540 CTCGCGGAAA CAAATGCTAA CTTGGATGAG ATAGTTGAGC TGGCAAAGCA ACTCCAGAGT 600 GAGACCAATA TAAAGCCATT ATGGGGAACT GCACAACTTT TCATGCATCC ACGTTACATG 660 CACGGAGCTG CTACTAGCCC AGAGGTCAAG GTGTATGCTT ATGCTGCTGC TCAAGTGAAG 720 AAAGCTTTGG AGGTTACTCA CTACCTAGGC GGTGAGAACT ACGTATTCTG GGGTGGAAGA 780 GAGGGTTACC AAACTCTTCT CAATACCGAT ATGAAGAGGG AACTTGAACA TTTGGCTAAC 840 TTTCTTCAAG CTGCTGTTAA CCACAAGAAG AAGATCGGCT TTAACGGAAC ATTGTTGATA 900 GAGCCTAAGC CACAAGAACC AACAAAGCAT CAGTATGACT GGGATGTTGC AACTACATTC 960 TCTTTCCTAC AGAAGTTTGG TCTTACAGGG GAATTCAAGA TAAATGTTGA GTGCAACCAT 1020 GCTACTCTCT CTGGACATAG CTGCCATCAC GAGCTTGAGA CTGCACGCAT TAATGACATT 1080 CTTGGAAACA TTGATGCAAA CACTGGTGAT CCACAGGTTG CTTGGGACAC GGATGAGTTC 1140 CTTACAGACA TTTCAGAAGC TACCTTGATT ATGTCAAGTG TAGTTAAGAA TGATGGACTT 1200 GCGCCTGGTG GCTTCAACTT TTACGCCAAA TTGCGGAGGG AGAGTACTGA TGTTGAGGAC 1260 CTGTTTATTG CCCATATCTC TGGGATGGAC ACCATGGCCC GCGGCCGCCG CAATGTTGTC 1320 AAGCTGATTG AGGATGGTTC CCTGGACGAG CTTGTACGCA AACGCTACCA GAGCTTTGAC 1380 ACTGAGATTG GTGCCATGAT CGAGGCTGGG AAGGGCGACT TTGAAACGCT AGAAAAGAAG 1440 GCCTTGGAGT GGGGCGAGCC AACCGTTCCA TCGGGCAAAC AGGAATTGGC TGAGATGCTG 1500 TTCCAATCCG CTCTGTAGAT GGCGGCCCAC GGTTCTAGGA ATAAAAAAGC AAGAGCGCGA 1560 CCTTGGAACG CCCAGCCGTC CTCGTCACTA CAGGCGATGT TCTATAGTTA GGCCTCCATG 1620 CAGTGAACCC TGTAAACAAA CTGCGTGGAG CTGAAAATAA TGTAACCTTA TATCAAAATT 1680 AAACTCGTTC TTCAACACGG AATTTGGCTT 1710 (2) INFORMATION FOR SEQ ID NO: 3 : (i) SEQUNCE CHARACTERISTICS: (A) LENGTH: 484 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION SEQ ID NO: 3: Met Lys Gly Gly Glu Leu Leu Val Leu Leu Leu Ala Ser Ser Leu Cys 1 * 5 10 15 Leu Ser Ala Ala Val Ala Ala Gin Glu Thr Cys Pro Ala Asp lie Gly 20 25 30 Ala Lys Lys Thr Asp Ala Ala Ser Asp Asp Trp Glu Gly Glu Phe Phe 35 40 45 Pro Gly lie Asp Lys lie Asn Tyr Glu Gly Pro Thr Ser Lys Lys Pro 50 55 60 Leu Ser Tyr Lys Trp Tyr Asn Ala Glu Glu Val lie Leu Gly Lys Lys 65 70 75 80 Met Lys Asp Trp Phe Arg Phe Ser Val Ala Phe Trp His Thr Phe Arg 85 90 95 Gly Thr Gly Gly Asp Pro Phe Gly Ala Pro Thr Lys Asn Trp Pro Trp 100 105 110 Glu Asp Gly Thr Asn Ser Leu Ala Met Ala Lys Arg Arg Met Lys Ala 115 120 125 His Phe Glu Phe Met Glu Lys Leu Gly Val Glu Arg Trp Cys Phe His 130 135 140 Asp Arg Asp lie Ala Pro Asp Gly Lys Thr Leu Ala Glu Thr Asn Ala 145 150 155 160 Asn Leu Asp Glu lie Val Glu Leu Ala Lys Gin Leu Gin Ser Glu Thr 165 170 175 Asn lie Lys Pro Leu Trp Gly Thr Ala Gin Leu Phe Met His Pro Arg 180 185 190 Tyr Met His Gly Ala Ala Thr Ser Pro Glu Val Lys Val Tyr Ala Tyr 195 200 205 Ala Ala Ala Gin Val Lys Lys Ala Leu Glu Val Thr His Tyr Leu Gly 210 215 220 Gly Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu Gly Tyr Gin Thr Leu 225 230 235 240 Leu Asn Thr Asp Met Lys Arg Glu Leu Glu His Leu Ala Asn Phe Leu 245 250 255 Gin Ala Ala Val Asn His Lys Lys Lys lie Gly Phe Asn Gly Thr Leu 260 265 270 Leu lie Glu Pro Lys Pro Gin Glu Pro Thr Lys His Gin Tyr Asp Trp 275 280 285 Asp Val Ala Thr Thr Phe Ser Phe Leu Gin Lys Phe Gly Leu Thr Gly 290 295 300 Glu Phe Lys lie Asn Val Glu Cys Asn His Ala Thr Leu Ser Gly His 305 310 315 320 Ser Cys His His Glu Leu Glu Thr Ala Arg lie Asn Asp lie Leu Gly 325 330 335 340 Asn lie Asp Ala Asn Thr Gly Asp Pro Gin Val Gly Trp Asp Thr Asp 345 350 355 Glu Phe Leu Thr Asp He Ser Glu Ala Thr Leu He Met Ser Ser Val 360 365 370 Val Lys Asn Asp Gly Leu Ala Pro Gly Gly Phe Asn Phe Tyr Ala Lys 375 380 385 Leu Arg Arg Glu Ser Thr Asp Val Glu Asp Leu Phe He Ala His He 390 395 400 405 Ser Gly Met Asp Thr Met Ala Arg Gly Arg Arg Arg Asn Val Lys Leu 410 415 420 He Glu Asp Gly Ser Leu Asp Glu Leu Val Arg Lys Arg Tyr Gin Ser 425 430 435 Phe Asp Thr Glu He Gly Ala Met He Glu Ala Gly Lys Gly Asp Phe 440 445 450 Glu Thr Leu Glu Lys Lys Ala Leu Glu Trp Gly Glu Pro Thr Val Pro 455 460 465 Ser Gly Lys Gin Glu Leu Ala Glu Met Leu Phe Gin Ser Ala Leu 470 475 480 484 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUNCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION SEQ ID NO: 4 : TTACTCGAGA ATTCATCGA 19 (2) INFORMATION FCR SEQ ID NO: 5: (i) SEQUNCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION SEQ ID NO: 5: TTATGGGGAA CTGCACAACT TTC 23 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION SEQ ID NO: 6: AAAAGGATCC ATGAAGGGCG GGGAGCTCCT GGTC 34 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi! SEQUENCE DESCRIPTION SEQ ID NO: 7: CTACGACAAG GTTAGGCGAG ACATCCCTAG GAATA 35 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (3) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (xi) SEQUENCE DESCRIPTION SEQ ID NO: 8: ATTAAGCCGG CGGGGAGCTC CTGGTC 36 (2) INFORMATION SEQUENCE SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (3) TYPE: aminohappo (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Phe Gly Leu Thr Gly Glu Phe Lys 1 5 (2) INFORMATION SEQUENCE SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: aminohappo (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Glu Gly Tyr Gin Thr Leu Leu Asn Thr Asp Met Lys 1 5 10 (2) INFORMATION SEQUENCE SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: aminohappo (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Tyr Met His Gly Ala Ala Thr Ser Pro Glu Val Lys 1 5 10 (2) INFORMATION SEQUENCE SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: aminohappo (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Ser Tyr Asn Ala Glu Glu Val He Leu Val Gly Lys 1 5 10 (2) INFORMATION SEQUENCE SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: aminohappo (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Trp Tyr Asn Ala Glu Glu Val He Leu Val Gly Lys 1 5 10 (2) INFORMATION SEQUENCE SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: aminohappo (C) STRANDEDNASS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Ala His Phe Glu Phe Met Glu Lys 1 5 (2) INFORMATION SEQUENCE SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: aminohappo (C) STRANDEDNeSS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Gly Thr Gly Gly Val Pro Phe Gly Ala Pro Thr Lys 1 5 10 (2) INFORMATION SEQUENCE SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: aminohappo (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Xxa Xxb Xxc Xxd Glu Leu Glu Thr Ala Arg 1 5 10 (2) INFORMATION SEQUENCE SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: aminohappo (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Met Lys Asp Xxx Leu Arg 1 5 (2) INFORMATION SEQUENCE SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: aminohappo (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Asn Asp Gly Leu Ala Pro Gly Gly Phe 1 5 (2) INFORMATION SEQUENCE SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: aminohappo (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: He Asn Tyr Glu Gly Pro Thr Ser Lys 1 5

Claims: CLAI MS 1. An isolated nucleic acid sequence which codes upon ex¬ pression in a procaryotic or eucaryotic host cell for a polypeptide having xylose isomerase activity, which nucleic acid sequence is selected from: a) the nucleic acid sequences shown in SEQ. ID. No. 1 and SEQ. ID. No. 2 or the complementary strands thereof; b) nucleic acid sequences which hybridise to the sequences defined in (a) above; c) nucleic acid sequences which, but for the degeneracy of the genetic code, would hybridise to the sequences defined in (a) or (b) above and which code for the same polypeptides as those defined in (a) or (b) above. 2. A nucleic acid sequence according to claim 1 that also codes for a signal sequence capable of directing the secretion of the said polypeptide in a host cell, said signal sequence being located 5' to the nucleic acid sequence defined in (a) (b) or (c) in claim 1 and in the same reading frame as it. 3. A polypeptide encoded by a nucleic acid sequence according to claim 1 or claim 2. 4. A polypeptide according to claim 3 comprising the sequence shown in SEQ. ID. No. 3. 5. A vector comprising a sequence according to claim 1 or claim 2 operably linked to a promoter capable of directing expression of the said sequence in a host cell. 6. A vector according to claim 5 wherein the promoter is capable of directing expression a yeast cell. 7. A vector according to claim 6 wherein the promoter is capable of directing expression in a cell of Saccharomyces cerevisiae or Schizosaccharomyces pombe. 8. Cells transformed or transfected with a vector accord¬ ing to any one of claims 5 to 7. 9. Cells according to claim 8 that are yeast cells. 10. Cells according to claim 9 that are cells of Saccharomy¬ ces cerevisiae or Schizosaccharomyces pombe. 11. A process of producing ethanol which comprises: (i) contacting cells according to any one of claims 8 to 10 with a substrate that comprises one or more carbon sources selected from xylose and polymerised xylose moieties; (ii) culturing the said cells in conditions under which the isomerisation of D-xylose to D-xylulose occurs and under which the D-xylulose is further catabolized to ethanol; and (iii) recovering the ethanol. 12. An antibody to a polypeptide of claim 3 or claim 4. 13. A process of producing a polypeptide according to claim 3 or claim 4 comprising expressing a nucleic acid sequence according to claim 1 in a cell according to any one of claims 8 to 10. 14. Ethanol produced by a process according to claim 10. 15. A process of producing xylulose by contacting a sub ¬ strate containing xylose with a polypeptide according to claim 2 or claim 3 or cells according to any one of claims 7 to 9; and recovering the thus produced xylulose.

Inventors: AHO SIRPA, KRISTO PAULA, SAARELAINEN RITVA, SUOMALAINEN IRMA, AHO, SIRPA, KRISTO, PAULO, SAARELAINEN, RITVA, SUOMALAINEN, IRMA
Applicants: AHO SIRPA, KRISTO PAULA, PRIMALCO LTD, SAARELAINEN RITVA, SUOMALAINEN IRMA, AHO, SIRPA, KRISTO, PAULO, SAARELAINEN, RITVA, SUOMALAINEN, IRMA
Application year: 1996
Main classification: C12N9
IPC codes: C12N9, C12N15, C12P7