Xylose isomerase with improved properties

• US 6,475,768 B1
• Filed: 04/07/2000
• Issued: 11/05/2002
• Est. Priority Date: 04/09/1999
• Status: Expired due to Fees

• Current Assignee: Scandinavian Technology Group AB
• Original Assignee: Forskarpatent i Syd AB (Government of Sweden)
• Inventors: Dackehag, Eva Anna Viktoria, van Zyl, Willem Heber, Gárdonyi, Márk, Otero, Ricardo Román Cordero, Hahn-Hägerdal, Bärbel
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US09/545,569
• Time in Patent Office: 942 Days
• Field of Search: 435/233, 435/320.1, 435/254.2, 435/161, 435/254.21, 435/254.22, 435/254.23, 536/23.2
• US Class Current: 435/233
• CPC Class Codes: C12N 9/92 Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
• CPC Class Codes: C12Y 503/01005 Xylose isomerase (5.3.1.5)

Abstract

The present invention relates to novel nucleic acid sequences which, upon expression in a procaryotic or eucaryotic cell, code for a polypeptide having an elevated specific xylose isomerase activity compared to the wildtype Thermus thermophilus xylose isomerase. They are selected from a) nucleic acid sequences shown in SEQ. ID. No 1; b) the complementary strand of the sequence defined in (a) above; c) nucleic acid sequences which hybridize to the sequences defined in (a) or (b) above; d) nucleic acid sequences which, but for the degeneracy of the genetic code, would hybridize to the sequences defined in (a), (b) or (c) above and which code for the same polypeptide as those defined in (a), (b) or (c) above. The present invention further provides a process of producing ethanol from xylose containing materials comprising contacting cells that express such nucleic acid sequences and novel modified xylose isomerases that advantageously can be applied in the production of fructose syrups.

Claims

1. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence encoded by SEQ. ID. No. 1,

wherein nnn at nucleotides 487-489 of SEQ. ID. No. 1 encodes any amino acid, and nnn at nucleotides 1114-1116 of SEQ. ID. No. 1 encodes any nucleotide except glutamic acid, and

wherein said polypeptide has at least 10% higher specific xylose isomerase activity than a xylose isomerase encoded by SEQ ID No. 2.

2. The isolated nucleic acid of claim 1, wherein said polypeptide has at least 10% higher specific xylose isomerase activity than the xylose isomerase encoded by SEQ. ID. No. 2 at mesophilic temperatures.

3. The isolated nucleic acid of claim 1, wherein said polypeptide has at least 10% higher specific xylose isomerase activity than the xylose isomerase encoded by SEQ. ID. No. 2 at temperatures above 80°

C.

4. The isolated nucleic acid of claim 1, wherein said polypeptide has at least 10% higher specific xylose isomerase activity than the xylose isomerase encoded by SEQ. ID. No. 2 at a pH lower than 7.5.

5. The isolated nucleic acid of claim 1, wherein said polypeptide has at least 10% higher specific xylose isomerase activity than the xylose isomerase encoded by SEQ. ID. No. 2 in the presence of xylitol.

6. The isolated nucleic acid of claim 1, wherein said nnn at nucleotides 487-489 of SEQ. ID. No. 1 encodes leucine.

7. The isolated nucleic acid of claim 1, wherein nnn at nucleotides 1114-1116 of SEQ. ID. No. 1 encodes glycine.

8. The isolated nucleic acid of claim 1, wherein nnn at nucleotides 487-489 of SEQ. ID. No. 1 encodes phenylalanine.

9. The isolated nucleic acid of claim 8, wherein said nnn at nucleotides 1114-1116 of SEQ. ID. No. 1 encodes glycine.

10. The isolated nucleic acid of claim 1, wherein nnn at nucleotides 487-489 of SEQ. ID. No. 1 encodes leucine.

11. The isolated nucleic acid of claim 10, wherein said nnn at nucleotides 1114-1116 of SEQ. ID. No. 1 encodes glycine.

12. The isolated nucleic acid of claim 1, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ. ID. No. 3, SEQ. ID. No. 5, SEQ. ID. No. 7, and SEQ. ID. No. 9.

13. The isolated nucleic acid of claim 1, wherein said nucleic acid is selected from the group consisting of DNA, RNA, and PNA.

14. The isolated nucleic acid of claim 1, wherein said nucleic acid is DNA.

22. A vector comprising the isolated nucleic acid of claim 1.

23. A cell comprising the vector of claim 22.

24. The cell of claim 23, which is a yeast cell.

25. The yeast cell of claim 24, wherein said yeast cell is from a species selected from the group consisting of Pichia, Candida, Schizosaccharomyces, Zygosaccharomyces, and Saccharomyces.

26. The yeast cell of claim 25, wherein said cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces carlsbergensis.

27. The yeast cell of claim 26, wherein said cell is a Saccharomyces cerevisiae.

28. A process for producing ethanol, wherein said process comprises:

a) contacting at least one cell of claim 23 with a substrate that contains one or more carbon sources selected from xylose and polymerized xylose moieties;

b) culturing said at least one cell under conditions where isomerization of D-xylose to D-xylulose occurs and the D-xylulose is catabolized to ethanol; and

c) optionally, recovering the ethanol.

29. A process for producing a polypeptide having an amino acid sequence comprising a sequence selected from the group consisting of SEQ. ID. No. 4, SEQ. ID. No. 6, SEQ. ID. No. 8, and SEQ. ID. No. 10, wherein said process comprises transcribing and translating at least a portion of the isolated nucleic acid of claim 1.

15. An isolated nucleic acid having a nucleotide sequence that is at least 85%, but less than 100%, identical to the nucleotide sequence of SEQ. ID. No. 2,

wherein said isolated nucleic acid encodes a polypeptide having xylose isomerase activity,

wherein said polypeptide has at least 10% higher specific xylose isomerase activity than the xylose isomerase encoded by SEQ. ID. No. 2, and

wherein said isolated nucleic acid is mutated, with respect to SEQ. ID. No. 2, such that nucleotide codon 1114-1116 encodes any amino acid except glutamic acid.

16. A vector comprising the isolated nucleic acid of claim 15.

17. A cell comprising the vector of claim 16.

18. The cell of claim 17, which is a yeast cell.

19. The yeast cell of claim 18, wherein said yeast cell is from a species selected from the group consisting of Pichia, Candida, Schizosaccharomyces, Zygosaccharomyces, and Saccharomyces.

20. The yeast cell of claim 19, wherein said cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, and Saccharomyces carlsbergensis.

21. The yeast cell of claim 20, wherein said cell is a Saccharomyces cerevisiae.