Nucleic acid molecule
AssigneeGlaxoSmithKline Australia Pty Limited
InventorsThilo Winzer, Ian Alexander Graham, Tracy Carol Walker
Life of this patent
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Abstract
The disclosure relates to a nucleic acid molecule isolated from a Papaver somniferum cultivar that produces the opiate alkaloid noscapine which comprises 10 genes involved in the biosynthesis of opiate alkaloids.
Claims42
An isolated nucleic acid molecule that comprises a gene cluster that encodes two or more polypeptides involved in the biosynthesis of opiate alkaloids or intermediates, wherein one of said two polypeptides is encoded by a nucleotide sequence selected from the group consisting of:
- i) a nucleotide sequence as set forth in SEQ ID NO:
- 8;
- ii) a nucleotide sequence, wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
- iii) a nucleic acid molecule, the complementary strand of which hybridizes under stringent hybridization conditions to the nucleotide sequence in SEQ ID NO:
- 8 and which encodes a polypeptide that has carboxylesterase activity;
- and
- iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:
- 18 or a nucleotide sequence that encodes a polypeptide that has 46% amino acid sequence identity across the full length amino acid sequence set forth in SEQ ID NO:
- 18 wherein said polypeptide has carboxylesterase activity.
An isolated nucleic acid molecule that comprises a gene cluster that encodes two or more polypeptides involved in the biosynthesis of opiate alkaloids or intermediates, wherein one of said two polypeptides is encoded by a nucleotide sequence selected from the group consisting of;
- i) a nucleotide sequence as set forth in SEQ ID NO:
- 9;
- ii) a nucleotide sequence, wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
- iii) a nucleic acid molecule, the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO:
- 9 and which encodes a polypeptide that has short-chain dehydrogenase/reductase activity;
- and
- iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:
- 19 or a nucleotide sequence that encodes a polypeptide that has is 46% amino acid sequence identity across the full length amino acid sequence set forth in SEQ ID NO:
- 19 wherein said polypeptide has short-chain dehydrogenase/reductase activity.
6-7. (canceled)
9-12. (canceled)
A nucleic acid molecule comprising a transcription cassette, wherein said cassette includes one or more nucleotide sequences shown in SEQ ID NO:
- 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and is adapted for expression by at least one promoter operably linked to said nucleotide sequence such that both sense and antisense molecules are transcribed from said cassette.
(canceled)
A method to produce a P. somniferum plant that has altered expression of a polypeptide comprising:
- i) mutagenizing a wild-type seed from a P. somniferum plant that does express said polypeptide;
- ii) cultivating the seed in i) to produce first and subsequent generations of plants;
- iii) obtaining seed from the first generation plant and subsequent generations of plants;
- iv) determining if the seed from said first and subsequent generations of plants has altered nucleotide sequence and/or altered expression of said polypeptide;
- v) obtaining a sample and analysing the nucleic acid sequence of a nucleic acid molecule selected from the group consisting of:
- a) a nucleic acid molecule comprising a nucleotide sequence as shown in SEQ ID NO:
- 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
- and
- b) a nucleic acid molecule that hybridises to the nucleic acid molecule in a) under stringent hybridisation conditions and that encodes a polypeptide with opiate alkaloid biosynthetic activity;
- and optionally
- vi) comparing the nucleotide sequence of the nucleic acid molecule in said sample to a nucleotide sequence of a nucleic acid molecule of the original wild-type plant.
(canceled)
An isolated polypeptide selected from the group consisting of:
- i) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:
- 17;
- ii) a polypeptide modified by addition deletion or substitution of at least one amino acid residue of the sequence presented in SEQ ID NO:
- 17 and which has retained or enhanced cytochrome P450 activity;
- iii) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:18;
- iv) a polypeptide modified by addition, deletion or substitution of at least one amino acid residue of the sequence presented in SEQ ID NO:
- 18 and which has retained or enhanced carboxylesterase activity;
- v) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:
- 19;
- vi) a polypeptide modified by addition, deletion or substitution of at least one amino acid residue of the sequence presented in SEQ ID NO:
- 19 and which has retained or enhanced short-chain dehydrogenase/reductase activity;
- vii) a polypeptide comprising-an amino acid sequence as shown in SEQ ID NO:
- 20;
- and
- viii) a polypeptide modified by addition, deletion or substitution of at least one amino acid residue of the sequence presented in SEQ ID NO:
- 20 and which has retained or enhanced acetyltransferase activity.
45-50. (canceled)
An isolated nucleic acid molecule that encodes a gene cluster of one or more polypeptides involved in the biosynthesis of opiate alkaloids or intermediates comprising or consisting of a nucleotide sequence selected from the group consisting of:
- i) a nucleotide sequence as represented by the sequence shown in SEQ ID NO:
- 7, 8, 9 or 10;
- ii) a nucleotide sequence, wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);
- iii) a nucleic acid molecule, the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO:
- 7, 8, 9 or 10 wherein said nucleic acid molecule encodes a polypeptide involved in the biosynthesis of P. somniferum opiate alkaloids or intermediates in the biosynthesis of opiate alkaloids;
- iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO:
- 17, 18, 19 or 20;
- and
- v) a nucleotide sequence that encodes a polypeptide modified by addition, deletion or substitution of at least one amino acid residue as represented in iv) above and which has retained or enhanced opiate alkaloid biosynthetic activity.
Dependencies are inferred from the claim text.