PUFA-PKS Genes From Ulkenia

• US 20090093033A1
• Filed: 04/08/2005
• Published: 04/09/2009
• Est. Priority Date: 04/08/2004
• Status: Active Grant

• Current Assignee: Nutrinova Nutrition Specialties & Food Ingredients GmbH (Celanese Corporation)
• Original Assignee: Nutrinova Nutrition Specialties & Food Ingredients GmbH (Celanese Corporation)
• Inventors: Rusing, Matthias, Kiy, Thomas, Luy, Markus
• Granted Patent: US 7,939,305 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Abstract

The invention relates to genes which are coded for sequences specific to polyketide synthases (PKS). The thus synthesized PKS is characterized by the enzymatic capacity thereof to produce PUFAs (polyunsaturated fatty acids). The invention also relates to the identification of the corresponding DNA-sequences, in addition to the use of said nucleotide sequences for the production of recombined and/or transgenic organisms.

Claims

1. PUFA-PKS, comprising at least one of the amino acid sequences represented in SEQ ID No. 6 (ORF 1), 7 (ORF 2), 8 and/or 80 (ORF 3) and have sequences homologous to them with at least 70% sequence homology that have the biological activity of at least one domain of the PUFA-PKS.

2. Isolated PUFA-PKS according to claim 1 with 10 or more ACP domains.

3. PUFA-PKS according to claim 1, characterized in that it comprises at least one amino acid sequence with at least 70% sequence homology with at least 500 directly successive amino acids of the sequences SEQ ID No. 6 (ORF 1), 7 (ORF 2) and 8 and/or 80 (ORF 3) and that has the biological activity of at least one domain of PUFA-PKS.

5. An isolated DNA molecule coding for an amino acid sequence according to claim 1 and DNA completely complementary with it.

6. The isolated DNA molecule according to claim 5, characterized in that it has at least 70% identity with at least 500 successive nucleotides from the SEQ ID No. 3, 4 and 5 and/or 9.

7. The DNA molecule according to claim 5, characterized in that it codes an amino acid sequence that is at least 70% homologous with at least 500 directly successive amino acids of the sequences SEQ ID No. 6 (ORF 1), 7 (ORF 2) and 8 and/or 80 (ORF 3).

8. A recombinant DNA molecule comprising one of the DNA molecules according to claim 5 functionally connected with at least one DNA sequence that controls the transcription, preferably selected from the group consisting of SEQ ID No. XX-YY (terminators/promoters) or parts thereof from at least 500 nucleotides as well as functional variants of them.

9. A recombinant host cell comprising a recombinant DNA molecule according to claim 8.

10. The recombinant host cell according to claim 9 that endogenically expresses the PUFA-PKS having at least one of the amino acid sequences represented in SEQ ID No. 6 (ORF 1), 7 (ORF 2), 8, 80 (ORF 3), 32, 34, 45, 58, 59, 60, 61, 72, 74 and/or 77 and have sequences homologous to them with at least 70% sequence homology and that have the biological activity of at least one domain of the PUFA-PKS, with the activity of at least one further domain of PUFA-PKS.

12. A method for producing oil containing PUFA, preferably DHA, comprising the cultivation of a host cell according to claim 9.

13. Oil, produced according to the method according to claim 12.

14. A method for producing biomass containing PUFA, preferably DHA, comprising the cultivation of a host cell according to claim 9.

15. Biomass, produced according to the method according to claim 14.

16. Recombinant biomass according to claim 15.

4. An amino acid sequence with at least 70% identity with at least 500 directly successive amino acids of the sequences SEQ ID No. 6 (ORF 1), 7 (ORF 2) and 8 and/or 80 (ORF 3) and that has the biological activity of at least one domain of PUFA-PKS.

22. An isolated DNA molecule coding for an amino acid sequence according to claim 4 and DNA completely complementary with it.

24. The isolated DNA molecule according to claim 4, characterized in that it has at least 70%-identity with at least 500 successive nucleotides from the SEQ ID No. 3, 4 and 5 and/or 9.

26. The DNA molecule according to claim 4 characterized in that it codes an amino acid sequence that is at least 70% homologous with at least 500 directly successive amino acids of the sequences SEQ ID No. 6 (ORF 1), 7 (ORF 2) and 8 and/or 80 (ORF 3).

28. A recombinant DNA molecule comprising one of the DNA molecules according to one of claims 22 functionally connected with at least one DNA sequence that controls the transcription, preferably selected from the group consisting of SEQ ID No. XX-YY (terminators/promoters) or parts thereof from at least 500 nucleotides as well as functional variants of them.

30. A recombinant host cell comprising a recombinant DNA molecule according to claim 28.

32. The recombinant host cell according to claim 30 that endogenically expresses the PUFA-PKS having at least one of the amino acid sequences represented in SEQ ID No. 6 (ORF 1), 7 (ORF 2), 8, 80 (ORF 3), 32, 34, 45, 58, 59, 60, 61, 72, 74 and/or 77 and have sequences homologous to them with at least 70% sequence homology and that have the biological activity of at least one domain of the PUFA-PKS, with the activity of at least one further domain of PUFA-PKS.

34. A method for producing oil containing PUFA, preferably DHA, comprising the cultivation of a host cell according to claim 30.

36. Oil, produced according to the method according to claim 34.

38. A method for producing biomass containing PUFA, preferably DHA, comprising the cultivation of a host cell according to claim 30.

40. Biomass, produced according to the method according to claim 38.

42. Recombinant biomass according to claim 40.

11. A recombinant host cell comprising a recombinant DNA construct in which the elements controlling the translation are selected from the group consisting of SEQ ID No. XX-YY (terminators/promoters) or parts thereof from at least 500 nucleotides as well as functional variants of them.

17. A method of producing artificial polyketides comprising the steps of using individual enzyme domains from the SEQ ID No. 6, 7, 8 and/or 80 comprising PUFA-PKS.

18. PUFA-PKS comprising at least one of the amino acid sequences represented in SEQ ID No. 32, 34, 45, 58, 59, 60, 61, 72, 74 and/or 77 and have sequences homologous to them with at least 70% sequence homology and that have the biological activity of at least one domain of the PUFA-PKS.

19. Isolated PUFA-PKS according to claim 18 with 10 or more ACP domains.

20. PUFA-PKS according to claim 18 characterized in that it comprises at least one amino acid sequence with at least 70% sequence homology with at least 500 directly successive amino acids of the sequences SEQ ID No. 6 (ORF 1), 7 (ORF 2) and 8 and/or 80 (ORF 3) and that has the biological activity of at least one domain of PUFA-PKS.

21. An isolated DNA molecule coding for an amino acid sequence according to claim 18 and DNA completely complementary with it.

23. The isolated DNA molecule according to claim 18, characterized in that it has at least 70%-identity with at least 500 successive nucleotides from the SEQ ID No. 3, 4 and 5 and/or 9.

25. The DNA molecule according to claim 18 characterized in that it codes an amino acid sequence that is at least 70% homologous with at least 500 directly successive amino acids of the sequences SEQ ID No. 6 (ORF 1), 7 (ORF 2) and 8 and/or 80 (ORF 3).

27. A recombinant DNA molecule comprising one of the DNA molecules according to one of claims 21 functionally connected with at least one DNA sequence that controls the transcription, preferably selected from the group consisting of SEQ ID No. XX-YY (terminators/promoters) or parts thereof from at least 500 nucleotides as well as functional variants of them.

29. A recombinant host cell comprising a recombinant DNA molecule according to claim 27.

31. The recombinant host cell according to claim 29 that endogenically expresses the PUFA-PKS having at least one of the amino acid sequences represented in SEQ ID No. 6 (ORF 1), 7 (ORF 2), 8, 80 (ORF 3), 32, 34, 45, 58, 59, 60, 61, 72, 74 and/or 77 and have sequences homologous to them with at least 70% sequence homology and that have the biological activity of at least one domain of the PUFA-PKS, with the activity of at least one further domain of PUFA-PKS.

33. A method for producing oil containing PUFA, preferably DHA, comprising the cultivation of a host cell according to claim 29.

35. Oil, produced according to the method according to claim 33.

37. A method for producing biomass containing PUFA, preferably DHA, comprising the cultivation of a host cell according to claim 29.

39. Biomass, produced according to the method according to claim 37.

41. Recombinant biomass according to claim 39.