OLIGONUCLEOTIDE SEQUENCE FOR USE IN PATHWAY ENGINEERING

Abstract

The present invention comprises a novel artificial oligonucleotide sequence which can initiate the transcription of a gene under various conditions at a high level. Further the invention relates to a recombinant DNA fragment comprising the artificial oligonucleotide sequence, an expression plasmid comprising the recombinant DNA fragment and a host cell transformed with the recombinant DNA fragment.

Claims

1. An oligonucleotide sequence comprising: at least 80% sequence identity to SEQ ID NO: 1, wherein said oligonucleotide increases the transcription rate of a RNA typed as messenger RNA fragment encoding for a protein selected from the group consisting of enzymes, structural proteins, coenzymes, transporters, antibodies, hormones and regulators, as regulatory RNA fragment, as enzymatically active RNA fragment or as transfer RNA fragment.

2. The oligonucleotide sequence of claim 1, said oligonucleotide sequence comprising at least 85% sequence identity to SEQ ID NO: 1.

3. The oligonucleotide sequence of claim 1, wherein the transcription rate of the RNA fragment in a yeast host cell is increased by at least 2-fold when growing the yeast host cell transformed with at least one recombinant DNA fragment comprising the oligonucleotide on at least two substrates selected from the group consisting of glucose, mannose, fructose, galactose, xylose, arabinose, sucrose, trehalose, raffinose, glycerol, ethanol, acetate and lactate.

4. The oligonucleotide sequence of claim 1, wherein the activity of an enzyme encoded by the RNA fragment controlled by the oligonucleotide in a yeast host cell is increased by at least 2-fold when growing the yeast host cell transformed with at least one recombinant DNA fragment comprising the oligonucleotide on at least two substrates selected from the group consisting of glucose, mannose, fructose, galactose, xylose, arabinose, sucrose, trehalose, raffinose, glycerol, ethanol, acetate and lactate.

5. The oligonucleotide sequence of claim 1, wherein the enzyme is a carbohydrate modifying enzyme.

6. The oligonucleotide sequence of claim 1, wherein the enzyme is a carbohydrate modifying enzyme selected from the group consisting of EC 5.1.3, EC 5.3.1, EC 2.7.1, EC 2.2.1, EC 2.2.1 and EC 1.1.1.

7. The oligonucleotide sequence of claim 1, wherein the protein is selected from the group of consisting of SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 20, SEQ ID No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 41, SEQ ID No: 42, SEQ ID No: 43, SEQ ID No: 44, SEQ ID No: 45, SEQ ID No: 46, SEQ ID No: 47, SEQ ID No: 48, SEQ ID No: 49, SEQ ID No: 50, SEQ ID No: 51, SEQ ID No: 52, and SEQ ID No: 53.

8. The oligonucleotide sequence of claim 1, wherein 1 to 80 nucleotides are substituted, deleted or inserted.

9. A recombinant DNA fragment comprising the oligonucleotide sequence of claim 1.

10. An expression plasmid comprising at least one recombinant DNA fragment of claim 9.

11. A host cell transformed with at least one recombinant DNA fragment of claim 9 or transformed with at least one expression plasmid of claim 10.

12. The oligonucleotide sequence of claim 1, said oligonucleotide sequence comprising at least 90% sequence identity to SEQ ID NO: 1.

13. The oligonucleotide sequence of claim 1, said oligonucleotide sequence comprising at least 95% sequence identity to SEQ ID NO: 1.

Description

EXAMPLES AND FIGURES

[0064] In the following the present invention is described by the examples and figures. The examples and figures are considered for illustrative purpose only and do not limit the scope of the present invention and claims in any respect.

Example 1: Growth Study—Selection of a Screening Strain

[0065] 5 different host cells have been transformed with a plasmid containing an exemplary oligonucleotide for testing purposes.

TABLE-US-00001 TABLE 1 list of tested host cells counter name origin strain A Ethanol Red ® Fermentis, France (Batch 897/1 production date Jan. 5, 2008) strain B Simi White ™ Lallemand, Canada (Lot: 02905340230601V) strain C Rhône 2226 ™ (Lot: 025556551030Y9) strain D CBS 7764 Centraalbureau voor Schimmelcultures Strain E CBS 6413 (Institute of the Royal Netherlands Academy of Arts and Sciences- KNAW), Netherland

[0066] The plasmid was constructed by recombination cloning in S. cerevisiae: A yeast cell was transformed with PCR products offered 45 bp overlap to each other. The fragments were yeast Marker (pUG6 87 to 1559 bp), E. coli Marker and Ori (pUG19 754 to 2534 bp), yeast Ori (S. cerevisiae S288C chromosome IV 44978 to 449831 and S. cerevisiae S288C chromosome II 63156 to 63454 bp) and the functional part (SEQ ID NO:10, SEQ ID NO:54, S. cerevisiae S288C chromosome XI 326407 to 326108 bp). Thereby the parts were flanked by the restriction sites SapI, SbfI, StuI and NotI, respectively.

[0067] The yeast strains were transformed with the re-isolated plasmid with the high-efficiency LiAc method according to Gietz and Schiestl.

[0068] The host cells have then been cultivated in 50 ml of a xylose-containing substrate (10 g/l yeast extract, 20 g/l pepton, 20 g/l Xylose+200 mg/l G418) under aerobic condition in 300 ml shake flask by 30° C. and 250 rpm. The results are shown in FIG. 1.

[0069] Strain B has been selected for further tests due to the excellent growth performance.

Example 2: Growth Study—Comparison of Different Plasmids Containing Different Oligonucleotides

[0070] Strain B has been transformed with 10 plasmids. The plasmids were constructed in the same way as described within example 1 harboring the oligonucleotides SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively.

[0071] Strain B was transformed with the plasmids as described within example 1 and cultivated under the same conditions. The results are shown in FIG. 2.

[0072] The host cell transformed by the plasmids containing an oligonucleotide according to the present invention (regulated by SEQ ID NOs:1 to 7) showed a significant higher growth performance than the host cell transformed by plasmids containing oligonucleotides of the state of the art SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:8.

Example 3: Transcript Level—Comparison of Different Plasmids Containing Different Oligonucleotides

[0073] The yeast strains harboring the different plasmids described in example 2 were cultivated in 100 ml of glucose-, mannose-, ethanol-, glycerol- or xylose-containing substrate (10 g/l yeast extract, 20 g/l pepton, 20 g/l carbon source+200 mg/l G418) in 500 ml shake flask by 30° C. and 250 rpm. 5 ml of the cultures were harvested at approximately OD.sub.600 2—centrifuged, washed with water and centrifuged (two times). After that the pellet of the culture was stored at −80° C.

[0074] The total RNA was extracted from the cells by using the RNeasy Mini Kit™, Qiagen Germany according to producer manual. Then that 500 ng RNA were translated into cDNA through the usage of the ThermoScript™ RT-PCR Kit, Life Technologies USA according to producer manual. By using the iQ™ SYBR® Green Supermix and the iQ™ iCycler, BIO RAD Germany, following the producer information, the concentration of ACT1 and XylA mRNA could by calculated by amplifying 225 and 236 bp tall PCR products.

[0075] The relative transcript levels (concentration of XylA RNA divided by the concentration of ACT1 RNA) are shown in FIG. 3.

TABLE-US-00002 TABLE 2 x-fold change of the XylA transcript level under the control of different oligonucleotides in comparison to the XylA transcript level under the control of SEQ ID NO: 9 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 Glucose 23.4 ± 5.7  23.9 ± 5.9  22.6 ± 5.8  24.4 ± 5.3  28.9 ± 5.1  26.4 ± 6.6  22.3 ± 4.5  Mannose 25.3 ± 6.5  27.6 ± 6.6  25.9 ± 7.0  23.3 ± 6.6  22.1 ± 4.0  22.4 ± 6.7  26.2 ± 7.3  Ethanol 4.6 ± 0.8 5.6 ± 1.1 3.9 ± 0.7 3.9 ± 0.8 4.5 ± 0.6 4.7 ± 0.6 4.9 ± 1.1 Glycerol 8.3 ± 2.2 7.6 ± 3.4 7.6 ± 2.7 9.3 ± 2.7 6.6 ± 1.3 6.8 ± 2.3 7.0 ± 2.3 Xylose 6.8 ± 3.3 6.6 ± 5.3 5.8 ± 3.2 8.4 ± 4.1 7.1 ± 4.6 7.7 ± 4.5 7.9 ± 4.4

[0076] The reporter system under the control of the oligonucleotides according to the present invention has shown a 4 to 29 fold increase of the transcript level. The transcript levels vary between the various growth conditions but a significant difference regarding the control of the oligonucleotide SEQ ID NO:1 to SEQ ID NO:7 was not visible.

Example 4: Enzyme Activity—Comparison of Different Plasmids Containing Different Oligonucleotides

[0077] 50 ml of the cultures as defined within example 3 were harvested at approximately OD.sub.600 2. Afterwards the pellet of the culture was stored at −80° C. In addition, a culture of strain B carrying the plasmid described within example 2 without the functional part (empty plasmid) was treated in the same way.

[0078] The thawed pellets were suspended in 400 μl buffer (100 mM Tris pH 7.5, 10 mM MgCl2) and homogenized. After the cell lysis the crude extracts were diluted to a total protein concentration of 1 μg/μl (measured by Bradford assay). The xylose isomerase activity assays were performed in 100 μl with 10% of the diluted crude extracts, 0.25 mM NADH, 3 U/ml sorbitol dehydrogenase and 500 mM Xylose. The reaction kinetics were followed photometrically at 340 nm.

[0079] The measured enzyme activities (minus background activity—empty plasmid) are shown in FIG. 4.

TABLE-US-00003 TABLE 3 x-fold change of the xylose isomerase activity under the control of different oligonucleotides in comparison to the xylose isomerase activity under the control of SEQ ID NO: 9 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 Glucose 22.5 ± 3.6 23.7 ± 3.4 24.8 ± 3.5 24.9 ± 4.0 23.9 ± 3.9 22.8 ± 4.1 20.6 ± 3.2 Mannose 20.4 ± 4.4 20.4 ± 4.5 18.6 ± 5.4 18.9 ± 4.5 19.7 ± 4.7 19.7 ± 5.3 18.6 ± 4.2 Ethanol 13.7 ± 2.2 13.0 ± 2.2 14.0 ± 2.3 15.2 ± 2.6 13.9 ± 2.3 14.6 ± 2.6 13.4 ± 2.7 Glycerol 14.8 ± 4.2 14.3 ± 3.8 16.9 ± 4.8 15.5 ± 4.3 15.4 ± 4.3 16.5 ± 4.6 15.3 ± 4.2 Xylose 14.3 ± 2.0 15.0 ± 2.5 16.7 ± 2.6 14.8 ± 2.4 14.8 ± 2.3 14.5 ± 2.2 14.5 ± 2.4

[0080] The reporter system under the control of the oligonucleotides according to the present invention has shown a 14 to 25 fold increase of the enzyme activity. Whereby the enzyme activities vary between the various growth conditions but a significant difference regarding the control of the oligonucleotide SEQ ID NO: 1 to SEQ ID NO: 7 was not visible.

BRIEF DESCRIPTION OF THE FIGURES

[0081] FIG. 1 shows the growth performance of 5 different yeast host cells (A to E) which have been transformed with a plasmid containing a gene encoding for SEQ ID NO:17 regulated by a oligonucleotide according to the state of art: SEQ ID NO:10.

[0082] FIG. 2 shows the growth performance of strain B transformed with different plasmids containing different oligonucleotides (oligonucleotides of the state of the art and oligonucleotides according to the present invention)

[0083] FIG. 3 shows a 4 to 29 fold increase of the transcript level of the reporter system under the control of the oligonucleotides according to the present invention (SEQ ID NOs 1 to 7) compared to the state of the art oligonucleotides SEQ ID NO 8, SEQ ID NO: 9 and SEQ ID NO: 10 on different substrates (glucose, mannose, ethanol, glycerol and xylose)

[0084] FIG. 4 shows a 14 to 25 fold increase of the enzyme activity of the reporter system under the control of the oligonucleotides according to the present invention (SEQ ID NOs 1 to 7) compared to the state of the art oligonucleotides SEQ ID NO 8, SEQ ID NO: 9 and SEQ ID NO: 10 on different substrates (glucose, mannose, ethanol, glycerol and xylose)

TABLE-US-00004 Sequence Listing Description: SEQ. ID NO: 1: synthetic oligonucleotide consisting of a combina- tion of SEQ. ID NO: 9 and SEQ. ID NO: 8 ccattctctg ctgctttgtt gcatcgtatc atcgcccgtt tcagccagtc ccgcataccc   60 cctttgcaac gttaacgtta ccgctagcgt ttaccatctc cacgctaaaa ggaaaacgaa  120 gattaagata aagttgggta aaatccgggg taagaggcaa gggggtagag aaaaaaaaac  180 cggagtcatt atatacgata ccgtccaggg taagacagtg atttctagct tccacttttt  240 tcaatttctt tttttcgttc caaatggcgt ccacccgtac atccggaatc tgacggcaca  300 agagccgatt agtggaagcc acggttacgt gattgcggtt tttttttcct acgtataacg  360 ctatgacggt agttgaatgt taaaaacgaa aacagagata ttgaattgac tcgtaggaac  420 aatttcgggc ccctgcgtgt tcttctgagg ttcatctttt acatttgctt ctgctggata  480 attttcagag gcaacaagga aaaattagat ggcaaaaagt cgtctttcaa ggaaaaatcc  540 ccaccatctt tcgagatccc ctgtaactta ttggcaactg aaagaatgaa aaggaggaaa  600 atacaaaata tactagaact gaaaaaaaaa aagtataaat agagacgata tatgccaata  660 cttcacaatg ttcgaatcta ttcttcattt gcagctattg taaaataata aaacatcaag  720 aacaaacaag ctcaacttgt cttttctaag aacaaagaat aaacacaaaa acaaaaagtt  780 tttttaattt taatcaaaaa   800 SEQ. ID NO: 2: synthetic oligonucleotide corresponding to SEQ. ID NO: 1 comprising the following mutations: g315a, c322g, a623-, t679c ccattctctg ctgctttgtt gcatcgtatc atcgcccgtt tcagccagtc ccgcataccc   60 cctttgcaac gttaacgtta ccgctagcgt ttaccatctc cacgctaaaa ggaaaacgaa  120 gattaagata aagttgggta aaatccgggg taagaggcaa gggggtagag aaaaaaaaac  180 cggagtcatt atatacgata ccgtccaggg taagacagtg atttctagct tccacttttt  240 tcaatttctt tttttcgttc caaatggcgt ccacccgtac atccggaatc tgacggcaca  300 agagccgatt agtgaaagcc agggttacgt gattgcggtt tttttttcct acgtataacg  360 ctatgacggt agttgaatgt taaaaacgaa aacagagata ttgaattgac tcgtaggaac  420 aatttcgggc ccctgcgtgt tcttctgagg ttcatctttt acatttgctt ctgctggata  480 attttcagag gcaacaagga aaaattagat ggcaaaaagt cgtctttcaa ggaaaaatcc  540 ccaccatctt tcgagatccc ctgtaactta ttggcaactg aaagaatgaa aaggaggaaa  600 atacaaaata tactagaact gaaaaaaaaa agtataaata gagacgatat atgccaatac  660 ttcacaatgt tcgaatccat tcttcatttg cagctattgt aaaataataa aacatcaaga  720 acaaacaagc tcaacttgtc ttttctaaga acaaagaata aacacaaaaa caaaaagttt  780 ttttaatttt aatcaaaaa  799 SEQ. ID NO: 3: synthetic oligonucleotide corresponding to SEQ. ID  NO: 1 comprising the following mutations:  t549c, a622-, a623-, c661a, t679c ccattctctg ctgctttgtt gcatcgtatc atcgcccgtt tcagccagtc ccgcataccc   60 cctttgcaac gttaacgtta ccgctagcgt ttaccatctc cacgctaaaa ggaaaacgaa  120 gattaagata aagttgggta aaatccgggg taagaggcaa gggggtagag aaaaaaaaac  180 cggagtcatt atatacgata ccgtccaggg taagacagtg atttctagct tccacttttt  240 tcaatttctt tttttcgttc caaatggcgt ccacccgtac atccggaatc tgacggcaca  300 agagccgatt agtggaagcc acggttacgt gattgcggtt tttttttcct acgtataacg  360 ctatgacggt agttgaatgt taaaaacgaa aacagagata ttgaattgac tcgtaggaac  420 aatttcgggc ccctgcgtgt tcttctgagg ttcatctttt acatttgctt ctgctggata  480 attttcagag gcaacaagga aaaattagat ggcaaaaagt cgtctttcaa ggaaaaatcc  540 ccaccatcct tcgagatccc ctgtaactta ttggcaactg aaagaatgaa aaggaggaaa  600 atacaaaata tactagaact gaaaaaaaaa gtataaatag agacgatata tgccaataat  660 tcacaatgtt cgaatccatt cttcatttgc agctattgta aaataataaa acatcaagaa  720 caaacaagct caacttgtct tttctaagaa caaagaataa acacaaaaac aaaaagtttt  780 tttaatttta atcaaaaa  798 SEQ. ID NO: 4: synthetic oligonucleotide corresponding to SEQ.  ID NO: 1 comprising the following mutations: c495a, g674a, t679c, a687g ccattctctg ctgctttgtt gcatcgtatc atcgcccgtt tcagccagtc ccgcataccc   60 cctttgcaac gttaacgtta ccgctagcgt ttaccatctc cacgctaaaa ggaaaacgaa  120 gattaagata aagttgggta aaatccgggg taagaggcaa gggggtagag aaaaaaaaac  180 cggagtcatt atatacgata ccgtccaggg taagacagtg atttctagct tccacttttt  240 tcaatttctt tttttcgttc caaatggcgt ccacccgtac atccggaatc tgacggcaca  300 agagccgatt agtggaagcc acggttacgt gattgcggtt tttttttcct acgtataacg  360 ctatgacggt agttgaatgt taaaaacgaa aacagagata ttgaattgac tcgtaggaac  420 aatttcgggc ccctgcgtgt tcttctgagg ttcatctttt acatttgctt ctgctggata  480 attttcagag gcaaaaagga aaaattagat ggcaaaaagt cgtctttcaa ggaaaaatcc  540 ccaccatctt tcgagatccc ctgtaactta ttggcaactg aaagaatgaa aaggaggaaa  600 atacaaaata tactagaact gaaaaaaaaa aagtataaat agagacgata tatgccaata  660 cttcacaatg ttcaaatcca ttcttcgttt gcagctattg taaaataata aaacatcaag  720 aacaaacaag ctcaacttgt cttttctaag aacaaagaat aaacacaaaa acaaaaagtt  780 tttttaattt taatcaaaaa  800 SEQ. ID NO: 5: synthetic oligonucleotide corresponding to SEQ. ID  NO: 1 comprising the following mutations:  a171-, c322g, c430t, c431t, c495a ccattctctg ctgctttgtt gcatcgtatc atcgcccgtt tcagccagtc ccgcataccc   60 cctttgcaac gttaacgtta ccgctagcgt ttaccatctc cacgctaaaa ggaaaacgaa  120 gattaagata aagttgggta aaatccgggg taagaggcaa gggggtagag aaaaaaaacc  180 ggagtcatta tatacgatac cgtccagggt aagacagtga tttctagctt ccactttttt  240 caatttcttt ttttcgttcc aaatggcgtc cacccgtaca tccggaatct gacggcacaa  300 gagccgatta gtggaagcca gggttacgtg attgcggttt ttttttccta cgtataacgc  360 tatgacggta gttgaatgtt aaaaacgaaa acagagatat tgaattgact cgtaggaaca  420 atttcgggtt cctgcgtgtt cttctgaggt tcatctttta catttgcttc tgctggataa  480 ttttcagagg caaaaaggaa aaattagatg gcaaaaagtc gtctttcaag gaaaaatccc  540 caccatcttt cgagatcccc tgtaacttat tggcaactga aagaatgaaa aggaggaaaa  600 tacaaaatat actagaactg aaaaaaaaaa agtataaata gagacgatat atgccaatac  660 ttcacaatgt tcgaatctat tcttcatttg cagctattgt aaaataataa aacatcaaga  720 acaaacaagc tcaacttgtc ttttctaaga acaaagaata aacacaaaaa caaaaagttt  780 ttttaatttt aatcaaaaa  799 SEQ. ID NO: 6: synthetic oligonucleotide corresponding to SEQ. ID  NO: 1 comprising the following mutations: c181a,  c205t, c322g, t549c, a623-, t679c ccattctctg ctgctttgtt gcatcgtatc atcgcccgtt tcagccagtc ccgcataccc   60 cctttgcaac gttaacgtta ccgctagcgt ttaccatctc cacgctaaaa ggaaaacgaa  120 gattaagata aagttgggta aaatccgggg taagaggcaa gggggtagag aaaaaaaaac  180 aggagtcatt atatacgata ccgttcaggg taagacagtg atttctagct tccacttttt  240 tcaatttctt tttttcgttc caaatggcgt ccacccgtac atccggaatc tgacggcaca  300 agagccgatt agtggaagcc agggttacgt gattgcggtt tttttttcct acgtataacg  360 ctatgacggt agttgaatgt taaaaacgaa aacagagata ttgaattgac tcgtaggaac  420 aatttcgggc ccctgcgtgt tcttctgagg ttcatctttt acatttgctt ctgctggata  480 attttcagag gcaacaagga aaaattagat ggcaaaaagt cgtctttcaa ggaaaaatcc  540 ccaccatcct tcgagatccc ctgtaactta ttggcaactg aaagaatgaa aaggaggaaa  600 atacaaaata tactagaact gaaaaaaaaa agtataaata gagacgatat atgccaatac  660 ttcacaatgt tcgaatccat tcttcatttg cagctattgt aaaataataa aacatcaaga  720 acaaacaagc tcaacttgtc ttttctaaga acaaagaata aacacaaaaa caaaaagttt  780 ttttaatttt aatcaaaaa  799 SEQ. ID NO: 7: synthetic oligonucleotide corresponding to SEQ.  ID NO: 1 comprising the following mutations: t186a, c322g,  c430t, c431t, a623-, t679c ccattctctg ctgctttgtt gcatcgtatc atcgcccgtt tcagccagtc ccgcataccc   60 cctttgcaac gttaacgtta ccgctagcgt ttaccatctc cacgctaaaa ggaaaacgaa  120 gattaagata aagttgggta aaatccgggg taagaggcaa gggggtagag aaaaaaaaac  180 cggagacatt atatacgata ccgtccaggg taagacagtg atttctagct tccacttttt  240 tcaatttctt tttttcgttc caaatggcgt ccacccgtac atccggaatc tgacggcaca  300 agagccgatt agtggaagcc agggttacgt gattgcggtt tttttttcct acgtataacg  360 ctatgacggt agttgaatgt taaaaacgaa aacagagata ttgaattgac tcgtaggaac  420 aatttcgggt tcctgcgtgt tcttctgagg ttcatctttt acatttgctt ctgctggata  480 attttcagag gcaacaagga aaaattagat ggcaaaaagt cgtctttcaa ggaaaaatcc  540 ccaccatctt tcgagatccc ctgtaactta ttggcaactg aaagaatgaa aaggaggaaa  600 atacaaaata tactagaact gaaaaaaaaa agtataaata gagacgatat atgccaatac  660 ttcacaatgt tcgaatccat tcttcatttg cagctattgt aaaataataa aacatcaaga  720 acaaacaagc tcaacttgtc ttttctaaga acaaagaata aacacaaaaa caaaaagttt  780 ttttaatttt aatcaaaaa   799 SEQ. ID NO: 8: oligonucleotide from Saccharomyces cereyisiae 5288C, upstream region of PFK2 SEQ. ID NO: 9: oligonucleotide from Saccharomyces cereyisiae 5288C, upstream region of HXT7 SEQ. ID NO: 10: oligonucleotide from Saccharomyces cereyisiae 5288C, upstream region of PGK1 SEQ. ID NO: 11: protein with aldose -1-epimerase activity from Saccharomyces cereyisiae EC1118, 104_6579 SEQ. ID NO: 12: protein with aldose -1-epimerase activity from Saccharomyces cerevisiae EC1119, 1F14_0056 SEQ. ID NO: 13: protein with aldose -1-epimerase activity from Saccharomyces cerevisiae S288C, YHR210C SEQ. ID NO: 14: protein with aldose -1-epimerase activity from  Saccharomyces cerevisiae x Saccharomyces kudriavzevii VI N7, YHR210C-like SEQ. ID NO: 15: protein with aldose -1-epimerase activity from Saccharomyces cerevisiae AWRI796, YHR210C-like SEQ. ID NO: 16: protein with aldose -1-epimerase activity from Lactococcus lactis, XylM SEQ. ID NO: 17: protein with xylose isomerase activity from Eubacterium saburreum, XylA SEQ. ID NO: 18: protein with xylose isomerase activity from Piromyces sp. E2, XylA SEQ. ID NO: 19: protein with xylose isomerase activity from Orpinomyces sp. ukk1, XylA SEQ. ID NO: 20: protein with xylose isomerase activity from Clostridium phytofermentans, XylA SEQ. ID NO: 21: protein with xylose isomerase activity from Ruminococcus flavefaciens, XylA SEQ. ID NO: 22: protein with xylose isomerase activity from Bacteroides uniformis, XylA SEQ. ID NO: 23: protein with xylose isomerase activity from Clostridium cellulolyticum, XylA SEQ. ID NO: 24: protein with xylose isomerase activity from Thermotoga maritima, XylA SEQ. ID NO: 25: protein with xylose isomerase activity from Bacillus stearothermophilus, XylA SEQ. ID NO: 26: protein with xylose isomerase activity from Bacteroides stercoris, XylA SEQ. ID NO: 27: protein with xylose isomerase activity from Parabacteroides distasonis, XylA SEQ. ID NO: 28: protein with xylose isomerase activity from Prevotella ruminicola, XylA SEQ. ID NO: 29: protein with xylose isomerase activity from Agrobacterium tumefaciens, XylA SEQ. ID NO: 30: protein with xylose isomerase activity from Clostridium cellulovorans, XylA SEQ. ID NO: 31: protein with xylose isomerase activity from Burkholderia cenocepacia, XylA SEQ. ID NO: 32: protein with xylose isomerase activity from Lactococcus lactis, XylA protein SEQ. ID NO: 33: protein with xylose isomerase activity from Thermoanaerobacter thermohydrosulfuricus, XylA SEQ. ID NO: 34: protein with xylose isomerase activity from Reticulitermes speratus, XylA protein, XylA SEQ. ID NO: 35: protein with xylose isomerase activity from uncultured bacteria from cow rumen, sequence number 2 within WO 2014 164392 SEQ. ID NO: 36: protein with xylose isomerase activity from uncultured bacteria from cow rumen, sequence number 1 within WO 2014 164392 SEQ. ID NO: 37: protein with xylose isomerase activity from Lachnospiraceae bacterium ICM7, XylA SEQ. ID NO: 38: protein with xylose isomerase activity from Lachnospiraceae bacterium oral taxon 107, XylA SEQ. ID NO: 39: protein with xylose isomerase activity from Lachnospiraceae bacterium oral taxon 082, XylA SEQ. ID NO: 40:  protein with xylose isomerase activity from uncultured bacterium, XYM1 from Parachin,N.S. and Gorwa-Grauslund, M.F. Isolation of xylose isomerases  by sequence- and function-based screening from a soil metagenome library,  Biotechnol Biofuels 4 (1), 9 (2011) SEQ. ID NO: 41: protein with xylose isomerase activity from uncultured bacterium, XYM2 from Parachin,N.S. and Gorwa-Grauslund, M.F. Isolation of xylose isomerases  by sequence- and function-based screening from a soil metagenome library,  Biotechnol Biofuels 4 (1), 9 (2011) SEQ. ID NO: 42: protein with xylose isomerase activity from Thermus thermophilus, XylA SEQ. ID NO: 43: protein with xylose isomerase activity from Escherichia coli, XylA SEQ. ID NO: 44: protein with xylulokinase activity from Saccharomyces cereyisiae 5288C, XKS1 SEQ. ID NO: 45: protein with xylulokinase activity from Scheffersomyces (Pichia) stipites CBS 6054, XKS1 SEQ. ID NO: 46: protein with xylulokinase activity from Trichoderma reesei QM6a, TRIREDRAFT_123288 SEQ. ID NO: 47: protein with transaldolase activity from Saccharomyces cereyisiae 5288C, TAL1 SEQ. ID NO: 48: protein with transaldolase activity from Saccharomyces cereyisiae 5288C, NQM1 SEQ. ID NO: 49: protein with transaldolase activity from Scheffersomyces (Pichia)  6054, TAL1 SEQ. ID NO: 50: protein with transketolase activity from Saccharomyces cereyisiae S288C, stipitis CBS TKL1 SEQ. ID NO: 51: protein with transketolase activity from Saccharomyces cereyisiae S288C, TKL2 SEQ. ID NO: 52: protein with transketolase activity from Scheffersomyces (Pichia)  stipitis CBS 6054, TKL1 SEQ. ID NO: 53: protein with aldcohol dehydrogenase activity from Saccharomyces  cereyisiae 5288C, ADH1 SEQ. ID NO: 54: synthetic DNA sequence encoding for SEQ. ID NO: 17 atgaaggaat tcttcccagg tatttctcca gttaagttcg aaggtagaga ctctaagaac   60 ccattgtctt tcaagtacta cgacgctaag agagttatta tgggtaagac catggaagaa  120 cacttgtctt tcgctatggc ttggtggcac aacttgtgtg cttgtggtgt tgacatgttc  180 ggtcaaggta ccgttgacaa gtctttcggt gaatcttctg gtaccatgga acacgctaga  240 gctaaggttg acgctggtat tgaattcatg aagaagttgg gtattaagta ctactgtttc  300 cacgacaccg acattgttcc agaagaccaa gaagacatta acgttaccaa cgctagattg  360 gacgaaatta ccgactacat tttggaaaag accaaggaca ccgacattaa gtgtttgtgg  420 accacctgta acatgttctc taacccaaga ttcatgaacg gtgctggttc ttctaactct  480 gctgacgttt tctgtttcgc tgctgctcaa gctaagaagg gtttggaaaa cgctgttaag  540 ttgggtgcta agggtttcgt tttctggggt ggtagagaag gttacgaaac cttgttgaac  600 accgacatga agttggaaga agaaaacatt gctaccttgt tcaccatgtg tagagactac  660 ggtagatcta ttggtttcat gggtgacttc tacattgaac caaagccaaa ggaaccaatg  720 aagcaccaat acgacttcga cgctgctacc gctattggtt tcttgagaaa gtacggtttg  780 gacaaggact tcaagttgaa cattgaagct aaccacgcta ccttggctgg tcacaccttc  840 caacacgaat tgagagtttg tgctgttaac ggtatgatgg gttctgttga cgctaaccaa  900 ggtgacacct tgttgggttg ggacaccgac caattcccaa ccaacgttta cgacaccacc  960 ttggctatgt acgaaatttt gaaggctggt ggtttgagag gtggtttgaa cttcgactct 1020 aagaacagaa gaccatctaa caccgctgac gacatgttct acggtttcat tgctggtatg 1080 gacaccttcg ctttgggttt gattaaggct gctgaaatta ttgaagacgg tagaattgac 1140 gacttcgtta aggaaagata cgcttcttac aactctggta ttggtaagaa gattagaaac 1200 agaaaggtta ccttgattga atgtgctgaa tacgctgcta agttgaagaa gccagaattg 1260 ccagaatctg gtagacaaga atacttggaa tctgttgtca acaacatttt gttcggtggt 1320 tctggttaa 1329