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Title:
COMPOSITIONS AND METHODS FOR PRODUCING STEVIOL AND STEVIOL GLYCOSIDES
Document Type and Number:
WIPO Patent Application WO/2015/051454
Kind Code:
A1
Abstract:
The present invention relates to nucleotide sequences encoding ent-kaurenoic acid 13- hydroxylase polypeptides and polypeptides having ent-kaurenoic acid 13-hydroxylase activity. The invention also relates to methods of producing steviol and steviol glycosides.

Inventors:
BRANDLE, Jim (17 Willington Avenue, London, Ontario N6A 3Y5, CA)
RICHMAN, Alex (18 Westmount Road, Guelph, Ontario N1H 5H2, CA)
Application Number:
CA2014/050970
Publication Date:
April 16, 2015
Filing Date:
October 07, 2014
Export Citation:
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Assignee:
VINELAND RESEARCH AND INNOVATION CENTRE (4890 Victoria Avenue North, P.O. Box 4000Vineland Station, Ontario L0R 2E0, CA)
International Classes:
C12N15/53; A01H5/00; C07H15/24; C07K19/00; C12N9/02; C12N15/113; C12N15/63; C12P7/40; C12P19/56
Domestic Patent References:
WO2005038033A22005-04-28
Foreign References:
CA2580429C2012-05-22
Other References:
DATABASE GENBANK DATABASE 15 November 2012 (2012-11-15), accession no. F064616
DATABASE GENBANK DATABASE 15 November 2012 (2012-11-15), accession no. N594505
Attorney, Agent or Firm:
MBM INTELLECTUAL PROPERTY LLP (275 Slater Street, 14th FloorOttawa, Ontario K1P 5H9, CA)
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Claims:
Claims

1 . A nucleic acid comprising the nucleotide sequence encoding an ent-kaurenoic acid 13-hydroxylase polypeptide comprising the following sequence:

MKFKKFSCTHEAMERTTVSCVVGVATILLFYIWKISNRLWFKPKKIEKFLRDQGLKG TSYKFIYGDMKEMAQTMHESRSKPMALTHDIAPRVTPFFHKSVTTFGKTCFTWMGT KPMVHICEPTMIREVLTNNTKYQKQRGGNPLSKLLAKGLADAEGAQWFKHRKIINPA FHMEKLKHMIPAFYVSCNEMIDKWEKILIQESSSEVDVWPYLQTFSSDVISRTAFGS SFEEGRKIFELQRELAKLTMKAGNSIYIPGSRFFPTKNNKRMKEMDQQVKSSIKSIIN KRVVAMKAGEARH DDLLGILLDSNYKEIKHHGNSNFGLSIEDIIEECKLFYFAGQETT GNMLVWTMILLGQHSEWQTRAREEVLHVFEDKKPNIDGLSHLKVISIIFNEVLRLYSP AAFLRRQIHEETKLGNLILPAGTLIQINALILHHDKEMWGEDANDFKPERFYEGVSKV TKGQAVYLPFGGGPRICIGQNFALLEAKMALAMILQRFSFDLSPSYSHSPHALPTLQ PQFGAHLILHKL (SEQ ID NO:2).

2. A nucleic acid encoding a polypeptide having ent-kaurenoic acid 13-hydroxylase activity and comprising a sequence having 55%, 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO:2.

3. A nucleic acid molecule comprising the following nucleotide sequence:

ATGAAATTCAAAAAGTTTTCTTGTACCCACGAAGCCATGGAGAGAACTACAGTTT CTTGTGTTGTTGGAGTTGCAACAATCTTGTTGTTTTACATATGGAAGATTTCAAAT CGGTTGTGGTTCAAACCAAAGAAGATTGAGAAATTTCTAAGAGATCAAGGACTC AAAGGTACCTCTTATAAATTCATTTACGGAGATATGAAAGAGATGGCACAAACGA TGCACGAATCCAGGTCTAAACCCATGGCTCTAACTCACGATATTGCTCCACGTG TCACGCCCTTCTTCCACAAATCCGTCACCACTTTTGGTAAGACATGTTTTACATG GATGGGAACAAAACCTATGGTACATATATGTGAACCTACTATGATACGAGAGGT TTTGACTAATAATACTAAATATCAAAAGCAAAGGGGAGGTAATCCATTATCAAAG TTGCTAGCTAAGGGATTAGCAGATGCGGAAGGTGCTCAATGGTTTAAACATAGA AAAATCATCAATCCTGCATTTCATATGGAGAAACTCAAGCATATGATACCAGCAT TTTATGTTAGTTGTAATGAGATGATTGACAAATGGGAGAAAATTTTAATACAAGAA AGCTCAAGTGAAGTGGATGTGTGGCCTTATCTTCAAACATTTTCAAGTGATGTAA TTTCACGTACAGCGTTTGGTAGTAGTTTTGAGGAAGGAAGAAAGATATTTGAACT ACAAAGGGAACTAGCAAAGCTTACAATGAAGGCTGGAAATTCAATTTACATTCCA GGATCAAGATTTTTTCCGACTAAAAACAATAAAAGGATGAAAGAAATGGACCAAC AAGTGAAAAGTTCAATAAAAAGCATAATTAATAAACGAGTCGTTGCGATGAAAGC TGGAGAAGCTAGGCATGATGATCTTTTGGGCATACTTTTGGATTCCAATTACAAA GAAATTAAACATCATGGGAATAGCAATTTCGGATTAAGCATCGAAGACATTATTG

AAGAATGTAAACTTTTCTACTTTGCAGGACAAGAGACCACTGGAAATATGCTTGT

TTGGACTATGATTTTGTTAGGCCAACACTCAGAATGGCAGACACGTGCTAGAGA

GGAAGTTTTGCATGTATTTGAAGATAAAAAACCAAATATTGATGGCTTAAGTCAT

CTAAAAGTGATTAGCATTATCTTTAATGAGGTTCTTAGACTATATTCACCAGCTG

CATTTCTTAGACGCCAAATACACGAGGAAACCAAATTAGGTAACTTGATATTACC

AGCAGGAACCCTCATCCAAATAAACGCGTTAATATTGCATCATGACAAAGAAATG

TGGGGTGAAGATGCAAATGATTTTAAACCTGAAAGATTTTATGAAGGTGTTTCAA

AGGTTACTAAAGGACAAGCTGTGTACCTTCCCTTTGGTGGCGGCCCACGTATAT

GCATTGGACAAAATTTTGCACTACTAGAAGCGAAAATGGCTCTTGCAATGATTCT

ACAACGCTTTTCATTTGATTTATCGCCTTCGTACTCGCATTCTCCGCATGCTTTA

CCTACTCTACAACCACAATTTGGTGCTCACTTG ATTTTACACAAACTCTAA (S EQ

ID N0:1 ).

4. A nucleic acid comprising a sequence which hybridizes to the complement of SEQ ID NO:1 under stringent hybridization conditions and encodes a polypeptide exhibiting ent-kaurenoic acid 13-hydroxylase activity.

5. The nucleic acid of claim 4, wherein the stringent hybridization conditions comprise hybridization in 4XSSC at 65 °C for 8-16 hours, followed by washing in 0.1 XSSC at 65 °C for one hour, or hybridization in 5XSSC and 50% formamide at 42 °C for 8-16 hours, followed by washing in about 0.5XSSC to about 0.2XSSC at 65 °C for one hour, wherein the nucleic acid encodes a polypeptide that exhibits ent-kaurenoic acid 13-hydroxylase activity.

6. The nucleic acid of claim 4 or 5 wherein said nucleotide sequence exhibits between about 52% and 100% sequence identity with SEQ ID NO: 1 .

7. A nucleic acid encoding a polypeptide exhibiting ent-kaurenoic acid 13-hydroxylase activity and comprising a nucleotide sequence 55%, 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99% identity to the sequence set forth in identity with SEQ ID NO:1 .

8. A construct comprising the nucleic acid of any one of claims 1 to 7.

9. The construct of claim 8, wherein said construct is an expression vector.

10. A polypeptide encoded by the nucleic acid of any one of claims 1 to 8.

1 1 . A polypeptide comprising an amino acid sequence defined by SEQ ID NO:2, or a biologically active fragment or variant thereof.

12. A polypeptide having ent-kaurenoic acid 13-hydroxylase activity and comprising a sequence having 55% to 100% identity to the sequence set forth in SEQ ID NO:2.

13. A fusion protein of SEQ ID NO:2, a fragment or variant thereof that exhibits ent- kaurenoic acid 13-hydroxylase activity and a heterologous amino acid sequence.

14. A fusion protein comprising the polypeptide of any one of claims 10 to 12 and a heterlogous amino acid sequence.

15. The fusion protein of claim 13 or 14, wherein said heterologous amino acid sequence is selected from the group consisting of a membrane targeting sequence, an organelle targeting sequence, a secretion signal sequence, a purification sequence and any combination thereof.

16. A cell comprising the nucleic acid of any one of claims 1 to 7, the construct of claim 8 or 9, expressing the protein of any one of claims 10 to 12 or expressing the fusion protein of any one of claims 14 to 15.

17. The cell of claim 16, wherein the cell is a prokaryotic cell.

18. The cell of claim 16, wherein the cell is an eukaryotic cell.

19. The cell of claim 18, wherein said eukaryotic cell is a plant cell, mammalian cell, yeast, fungi or insect cell.

20. A microorganism comprising the nucleic acid of any one of claims 1 to 7, the

construct of claim 8 or 9, expressing the polypeptide of any one of claims 10 to 12 or expressing the fusion protein of any one of claims 14 to 15.

21 . A plant comprising the nucleic acid of any one of claims 1 to 7, the construct of claim 8 or 9, expressing the polypeptide of any one of claims 10 to 12 or expressing the fusion protein of any one of claims 14 to 15.

22. A method of producing a polypeptide having ent-kaurenoic acid 13-hydroxylase activity comprising culturing the cell according to any one of claims 16 to 19 or the microorganism of claim 20.

23. A method of producing a polypeptide having ent-kaurenoic acid 13-hydroxylase activity, said method comprising,

a) providing a host comprising the nucleic acid of any one of claims 1 to 7, and; b) expressing said nucleic acid in the host and thereby producing said polypeptide having ent-kaurenoic acid 13-hydroxylase activity.

24. The method of claim 23, wherein said host is a plant cell.

25. The method of claim 23, wherein said host is a microorganism.

26. The method of claim 25, wherein said microorganism is a bacterium, yeast or fungi.

27. A process for producing a host capable of expressing a polypeptide having ent- kaurenoic acid 13-hydroxylase activity comprising genetically engineering said host with the nucleic acid of any one of claims 1 to 7, or with the construct of claim 8 or 9.

28. A method of producing steviol in a host comprising,

a) selecting a host that produces ent-kaurenoic acid;

b) genetically engineering the host with a nucleotide sequence encoding a polypeptide having ent-kaurenoic acid 13-hydroxylase activity;and

c) expressing the polypeptide having ent-kaurenoic acid 13-hydroxylase in the host to convert ent-kaurenoic acid to steviol.

29. A method of producing steviol in a host comprising,

a) selecting a host that produces ent-kaurenoic acid;

b) transforming said host with the nucleic acid of any one of claims 1 to 7, and; c) expressing said nucleic acid to produce a polypeptide having ent-kaurenoic acid 13-hydroxylase in the host to convert ent-kaurenoic acid to steviol.

30. A method of producing a steviol glycoside in a host comprising, a) selecting a host that produces ent-kaurenoic acid; b) transforming the host with a first nucleotide sequence encoding a polypeptide having ent-kaurenoic acid 13-hydroxylase activity, and at least one other nucleotide sequence encoding one or more glucosyltransferases to catalyse the addition of one or more glucose molecules to steviol, or glucosyl-steviol; c) expressing the polypeptide having ent-kaurenoic acid 13-hydroxylase and said one or more glucosyltransferases in the host to convert ent- kaurenoic acid to one or more steviol glycosides.

31 . A method of producing a steviol glycoside in a host comprising,

a) selecting a host that produces ent-kaurenoic acid;

b) genetically engineering said plant or plant cell with the nucleic acid of any one of claims 1 to 7, and at least one other nucleic acid comprising a sequence encoding one or more glucosyltransferases to catalyse the addition of one or more glucose molecules to steviol, or glucosyl-steviol;

c) expressing said polypeptide having ent-kaurenoic acid 13-hydroxylase and said one or more glucosyltransferases in the cell to convert ent-kaurenoic acid to one or more steviol glycosides.

32. The method of claim 31 , wherein said steviol glycoside comprises stevioside,

rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside, rubusoside, steviolmonoside, steviolbioside, 19-Ο-β glucopyranol-steviol, rebaudioside M, rebaudioside N, rebaudioside 0, rebaudioside G, 19-0-p-(2-1 )-steviolbioside, 19-0-p-(3-1 )-steviolbioside, 19-0-β-(2-1 ,3-1 )- stevioltrioside or any combination thereof.

33. The method of any one of claims 28 to 32, wherein said host is a microorganism.

34. The method of claim 33, wherein said microorganism is a bacterium, yeast or fungi.

35. The method of any one of claims 28 to 32, wherein host is a plant, plant cell, insect cell or mammalian cell.

36. The method of claim 35, wherein said plant or plant cell is Arabidopsis, tobacco, sunflower, Stevia rebaudiana, Thlapsi arvense or a member of the Cruciferae family.

37. A method of producing a steviol glycoside in a plant or plant cell comprising,

a) selecting a plant or plant cell that produces enf-kaurenoic acid;

b) transforming said plant or plant cell with the nucleic acid of any one of claims 1 to 7, and a nucleic acid comprising a sequence that downregulates the expression of enf-kaurenoic acid 13 oxidase;

c) expressing said nucleic acids and recovering said steviol glycoside.

38. An in-vitro method of producing steviol or one or more steviol glycosides comprising, a) reacting ent-kaurenoic acid with a polypeptide having ent-kaurenoic acid 13- hydroxylase activity under conditions to produce steviol, and; b) optionally reacting said steviol with one or more glucosyltransferases under conditions to produce one or more steviol glycosides.

39. A nucleic acid comprising a sequence complementary to the sequence of the nucleic acid of any one of claims 1 to 7.

Description:
Compositions and Methods for Producing Steviol and Steviol Glycosides FIELD OF INVENTION

The present invention relates to compositions and methods for producing sweeteners. More specifically, the present invention relates to compositions and methods for producing steviol and steviol glycosides.

BACKGROUND OF THE INVENTION

The worldwide demand for high potency sweeteners is increasing. The sweet herb of Paraguay, Stevia rebaudiana Bertoni, produces a high potency sweetener. The sweet steviol glycosides accumulate in Stevia leaves where they may comprise from 10 to 20% of the leaf dry weight. Stevioside and rebaudioside A are both heat and pH stable (Chang and Cook, 1983, J. Agric. Food Chem. 31 (2), pp 409-412), and suitable for use in carbonated beverages and many other foods. Stevioside is between 1 10 and 270 times sweeter than sucrose, rebaudioside A between 150 and 320 times sweeter than sucrose.

Early steps in steviol biosynthesis involve the plastid localized 1 -deoxy-D-xylulose 5- phosphate (DXP) pathway, resulting in the formation of DXP from pyruvate and

glyceraldehyde 3-phosphate by thiamine phosphate dependant DXP synthase (Totte et al. 2000, Can. J. Bot. 81 :517-522), and leading ultimately to the synthesis of geranyl geranyl diphosphate (GGDP). Like all diterpenes, steviol is synthesized from GGDP, first by protonation-initiated cyclization to (-)-copalyl diphosphate (CDP) by CDP synthase (CPS) (Richman et al., 1999, Plant J. 19:41 1 -421 ). Next, (-)-kaurene is produced from CDP by an ionization-dependent cyclization catalysed by (-)-kaurene synthase (KS). KS polypeptides are known in the art (see, for example, GenBank Accession Numbers AAD34295;

AAD34294; NP_001 105096; XP_00231 1286 and AF034774 ). (-)-Kaurene is then oxidized at the C-19 position to (-)-kaurenoic acid, by kaurene oxidase (KO). KO polypeptides are also known in the art (see, for example GenBank Accession Numbers ABA42921 ;

AAC39505; CAA76703 and BAB59027). Steviol is produced by the hydroxylation of (-)- kaurenoic acid at the C-13 positionby kaurenoic acid 13-hydroxylase (see, for example, Genbank Accession Numbers ACD93722; NP_197872; XP_002282091 and ABC59076. Steviol glucosides are formed by four glycosylation reactions that start with steviol and end with rebaudoside A (Richman et al. 2005, Plant J. 41 :56-67). The steps involve the addition of glucose to the C-13 hydroxyl, the transfer of glucose to the C-2' and C-3' of the 13-0- glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl. l SUMMARY OF THE INVENTION

An object of the present invention provides compositions and methods for producing steviol and steviol glycosides. In accordance with an aspect of the present invention, there is provided a nucleic acid comprising the nucleotide sequence encoding an ent-kaurenoic acid 13-hydroxylase polypeptide comprising the following sequence:

MKFKKFSCTHEAMERTTVSCVVGVATILLFYIWKISNRLWFKPKKIEKFLRDQ

GLKGTSYKFIYGDMKEMAQTMH ESRSKPMALTHDIAPRVTPFFHKSVTTFG

KTCFTWMGTKPMVHICEPTMIREVLTNNTKYQKQRGGNPLSKLLAKGLADA

EGAQWFKHRKIINPAFHMEKLKHMIPAFYVSCNEMIDKWEKILIQESSSEVD

VWPYLQTFSSDVISRTAFGSSFEEGRKIFELQRELAKLTMKAGNSIYIPGSRF

FPTKNNKRMKEMDQQVKSSIKSIINKRVVAMKAGEARHDDLLGILLDSNYKEI

KHHGNSNFGLSIEDIIEECKLFYFAGQETTGNMLVWTMILLGQHSEWQTRA

REEVLHVFEDKKPNIDGLSHLKVISIIFNEVLRLYSPAAFLRRQIHEETKLGNLI

LPAGTLIQINALILHHDKEMWGEDANDFKPERFYEGVSKVTKGQAVYLPFGG

GPRICIGQNFALLEAKMALAMILQRFSFDLSPSYSHSPHALPTLQPQFGAHLI

LHKL (SEQ ID NO:2).

In accordance with another aspect of the present invention, there is provided a nucleic acid encoding a polypeptide having ent-kaurenoic acid 13-hydroxylase activity and comprising a sequence having 55%, 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99% identity to the sequence set forth in SEQ ID NO:2.

In accordance with another aspect of the present invention, there is provided a nucleic acid molecule comprising the following nucleotide sequence:

ATGAAATTCAAAAAGTTTTCTTGTACCCACGAAGCCATGGAGAGAACTAC

AGTTTCTTGTGTTGTTGGAGTTGCAACAATCTTGTTGTTTTACATATGGAA

GATTTCAAATCGGTTGTGGTTCAAACCAAAGAAGATTGAGAAATTTCTAA

GAGATCAAGGACTCAAAGGTACCTCTTATAAATTCATTTACGGAGATATG

AAAGAGATGGCACAAACGATGCACGAATCCAGGTCTAAACCCATGGCTC

TAACTCACGATATTGCTCCACGTGTCACGCCCTTCTTCCACAAATCCGTC

ACCACTTTTGGTAAGACATGTTTTACATGGATGGGAACAAAACCTATGGT

ACATATATGTGAACCTACTATGATACGAGAGGTTTTGACTAATAATACTAA ATATCAAAAGCAAAGGGGAGGTAATCCATTATCAAAGTTGCTAGCTAAGG

GATTAGCAGATGCGGAAGGTGCTCAATGGTTTAAACATAGAAAAATCATC

AATCCTGCATTTCATATGGAGAAACTCAAGCATATGATACCAGCATTTTAT

GTTAGTTGTAATGAGATGATTGACAAATGGGAGAAAATTTTAATACAAGA

AAGCTCAAGTGAAGTGGATGTGTGGCCTTATCTTCAAACATTTTCAAGTG

ATGTAATTTCACGTACAGCGTTTGGTAGTAGTTTTGAGGAAGGAAGAAAG

ATATTTGAACTACAAAGGGAACTAGCAAAGCTTACAATGAAGGCTGGAAA

TTCAATTTACATTCCAGGATCAAGATTTTTTCCGACTAAAAACAATAAAAG

GATGAAAGAAATGGACCAACAAGTGAAAAGTTCAATAAAAAGCATAATTA

ATAAACGAGTCGTTGCGATGAAAGCTGGAGAAGCTAGGCATGATGATCT

TTTGGGCATACTTTTGGATTCCAATTACAAAGAAATTAAACATCATGGGA

ATAGCAATTTCGGATTAAGCATCGAAGACATTATTGAAGAATGTAAACTTT

TCTACTTTGCAGGACAAGAGACCACTGGAAATATGCTTGTTTGGACTATG

ATTTTGTTAGGCCAACACTCAGAATGGCAGACACGTGCTAGAGAGGAAG

TTTTGCATGTATTTGAAGATAAAAAACCAAATATTGATGGCTTAAGTCATC

TAAAAGTGATTAGCATTATCTTTAATGAGGTTCTTAGACTATATTCACCAG

CTGCATTTCTTAGACGCCAAATACACGAGGAAACCAAATTAGGTAACTTG

ATATTACCAGCAGGAACCCTCATCCAAATAAACGCGTTAATATTGCATCA

TGACAAAGAAATGTGGGGTGAAGATGCAAATGATTTTAAACCTGAAAGAT

TTTATGAAGGTGTTTCAAAGGTTACTAAAGGACAAGCTGTGTACCTTCCC

TTTGGTGGCGGCCCACGTATATGCATTGGACAAAATTTTGCACTACTAGA

AGCGAAAATGGCTCTTGCAATGATTCTACAACGCTTTTCATTTGATTTATC

GCCTTCGTACTCGCATTCTCCGCATGCTTTACCTACTCTACAACCACAAT

TTGGTGCTCACTTGATTTTACACAAACTCTAA (SEQ ID N0:1 ).

In accordance with another aspect of the present invention, there is provided a nucleic acid comprising a sequence which hybridizes to the complement of SEQ ID NO:1 under stringent hybridization conditions and encodes a polypeptide exhibiting ent-kaurenoic acid 13- hydroxylase activity.

In accordance with another aspect of the present invention, there is provided a nucleic acid encoding a polypeptide exhibiting ent-kaurenoic acid 13-hydroxylase activity and comprising a nucleotide sequence 55%, 60%, 70%, 75%, 80%, 90%, 95%, 98% or 99% identity to the sequence set forth in identity with SEQ ID NO:1 . In accordance with another aspect of the present invention, there is provided a polypeptide comprising an amino acid sequence defined by SEQ ID NO:2, or a biologically active fragment or variant thereof.

In accordance with another aspect of the present invention, there is provided a polypeptide having ent-kaurenoic acid 13-hydroxylase activity and comprising a sequence having 55% to 100% identity to the sequence set forth in SEQ ID NO:2.

In accordance with another aspect of the present invention, there is provided a fusion protein of SEQ ID NO:2, a fragment or variant thereof that exhibits ent-kaurenoic acid 13- hydroxylase activity and a heterologous amino acid sequence.

In accordance with another aspect of the present invention, there is provided a method of producing steviol in a host comprising: a) selecting a host that produces ent-kaurenoic acid; b) genetically engineering the host with a nucleotide sequence encoding a polypeptide having ent-kaurenoic acid 13-hydroxylase activity; and c) expressing the polypeptide having ent-kaurenoic acid 13-hydroxylase in the host to convert ent-kaurenoic acid to steviol.

In accordance with another aspect of the present invention, there is provided a method of producing steviol in a host comprising: a) selecting a host that produces ent-kaurenoic acid and has been genetically engineered with a nucleic acid encoding a polypeptide having ent- kaurenoic acid 13-hydroxylase activity; and b) expressing the polypeptide having ent- kaurenoic acid 13-hydroxylase in the host to convert ent-kaurenoic acid to steviol.

In accordance with another aspect of the present invention, there is provided a method of producing a steviol glycoside in a host comprising, a) selecting a host that produces ent- kaurenoic acid; b) genetically engineering the host with a first nucleic acid encoding a polypeptide having ent-kaurenoic acid 13-hydroxylase activity, and at least one other nucleic acid encoding one or more glucosyltransferases to catalyse the addition of one or more glucose molecules to steviol, or glucosyl-steviol; c) expressing the polypeptide having ent- kaurenoic acid 13-hydroxylase and said one or more glucosyltransferases in the host to convert ent-kaurenoic acid to one or more steviol glycosides. In accordance with another aspect of the present invention, there is provided an in-vitro method of producing steviol or one or more steviol glycosides comprising, a) reacting ent- kaurenoic acid with a polypeptide having ent-kaurenoic acid 13-hydroxylase activity under conditions to produce steviol, and; b) optionally reacting said steviol with one or more glucosyltransferases under conditions to produce one or more steviol glycosides.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawing wherein:

FIG. 1 shows a diagrammatic representation of biochemical pathways for the conversion of geranylgeranyl diphosphate to steviol and various steviol glycosides.

DETAILED DESCRIPTION

The present invention relates to compositions and methods for producing sweeteners. More specifically, the present invention relates to compositions and methods for producing steviol and steviol glycosides. As detailed above, steviol is produced by the hydroxylation of (-)- kaurenoic acid at the C-13 position. Ent-kaurenoic acid 13-hydroxylase activity catalyzes the conversion of ent-kaurenoic acid to steviol by mono-oxygenation. Accordingly, the present invention relates to polypeptides having ent-kaurenoic acid 13-hydroxylase activity, nucleic acids encoding such polypeptides, cells and plants expressing these polypeptides and methods which utilize the polynucleotides, polypeptides, cells and/or plants.

Definitions:

By the term "steviol" it is meant the diterpenoic compound hydroxy-ent-kaur-16-en-13-ol-19- oic acid, which is the hydroxylated form of the compound termed "ent-kaurenoic acid", which is ent-kaur-16-en-19-oic acid (see FIG. 1 ).

By the term "steviol glycoside" it is meant any of the glycosides of the aglycone steviol including, but not limited to stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudisode E, rebaudisode F, dulcoside, rubusoside, steviolmonoside, steviolbioside, 19-Ο-β glucopyranosol-steviol, rebaudioside M, rebaudioside N, rebaudioside 0, rebaudioside G, 19-0-p-(2-1 )-steviolbioside, 19-0-p-(3-1 )-steviolbioside and 19-0-β-(2- 1 ,3-1 )-stevioltrioside. By ent-kaurenoic acid 13-hydroxylase activity it is meant the activity associated with a polypeptide, either a full length or a fragment, that is capable of catalyzing or partially catalyzing the conversion of ent-kaurenoic acid to steviol by mono-oxygenation. In some embodiments, the polypeptide is ent-kaurenoic acid 13-hydroxylase, or a fragment thereof that is capable of catalyzing or partially catalyzing the conversion of ent-kaurenoic acid to steviol by mono-oxygenation.

By "operatively linked" it is meant that the particular sequences interact either directly or indirectly to carry out an intended function, such as mediation or modulation of gene expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences.

Nucleic Acids

According to an embodiment of the present invention, there is provided a nucleic acid encoding a polypeptide having ent-kaurenoic acid hydroxylase activity. In embodiments of the present invention, there is provided a nucleic acid comprising the nucleotide sequence comprising a sequence encoding the sequence as set forth in SEQ ID NO: 2 or a fragment or variant thereof. In certain embodiments, the fragment or variant of SEQ ID NO:2 has ent- kaurenoic acid hydroxylase activity. In further embodiments, there is provided a nucleic acid encoding a polypeptide having ent-kaurenoic acid 13-hydroxylase activity and comprising a sequence having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the sequence as set forth in SEQ ID NO:2.

SEQ ID NO:2:

MKFKKFSCTHEAMERTTVSCVVGVATILLFYIWKISNRLWFKPKKIEKFLRDQGLKGTSY

KFIYGDMKEMAQTMHESRSKPMALTHDIAPRVTPFFHKSVTTFGKTCFTWMGTKPMV HIC

EPTMIREVLTNNTKYQKQRGGNPLSKLLAKGLADAEGAQWFKHRKIINPAFHMEKLK HMI

PAFYVSCNEMIDKWEKILIQESSSEVDVWPYLQTFSSDVISRTAFGSSFEEGRKIFE LQR

ELAKLTMKAGNSIYIPGSRFFPTKNNKRMKEMDQQVKSSIKSIINKRVVAMKAGEAR HDD

LLGILLDSNYKEIKHHGNSNFGLSIEDIIEECKLFYFAGQETTGNMLVWTMILLGQH SEW

QTRAREEVLHVFEDKKPNIDGLSHLKVISIIFNEVLRLYSPAAFLRRQIHEETKLGN LIL

PAGTLIQINALILHHDKEMWGEDANDFKPERFYEGVSKVTKGQAVYLPFGGGPRICI GQN

FALLEAKMALAMILQRFSFDLSPSYSHSPHALPTLQPQFGAHLILHKL According to another embodiment of the present invention, there is provided a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 set forth below, a fragment or variant thereof. In some embodiments, the fragment or variant thereof encodes a polypeptide exhibiting ent-kaurenoic acid 13-hydroxylase activity.

SEQ ID NO:1 :

ATGAAATTCAAAAAGTTTTCTTGTACCCACGAAGCCATGGAGAGAACTACAGTTTCTTGT

GTTGTTGGAGTTGCAACAATCTTGTTGTTTTACATATGGAAGATTTCAAATCGGTTG TGG

TTCAAACCAAAGAAGATTGAGAAATTTCTAAGAGATCAAGGACTCAAAGGTACCTCT TAT

AAATTCATTTACGGAGATATGAAAGAGATGGCACAAACGATGCACGAATCCAGGTCT AA

ACCCATGGCTCTAACTCACGATATTGCTCCACGTGTCACGCCCTTCTTCCACAAATC CG

TCACCACTTTTGGTAAGACATGTTTTACATGGATGGGAACAAAACCTATGGTACATA TAT

GTGAACCTACTATGATACGAGAGGTTTTGACTAATAATACTAAATATCAAAAGCAAA GGG

GAGGTAATCCATTATCAAAGTTGCTAGCTAAGGGATTAGCAGATGCGGAAGGTGCTC AA

TGGTTTAAACATAGAAAAATCATCAATCCTGCATTTCATATGGAGAAACTCAAGCAT ATG

ATACCAGCATTTTATGTTAGTTGTAATGAGATGATTGACAAATGGGAGAAAATTTTA ATA

CAAGAAAGCTCAAGTGAAGTGGATGTGTGGCCTTATCTTCAAACATTTTCAAGTGAT GT

AATTTCACGTACAGCGTTTGGTAGTAGTTTTGAGGAAGGAAGAAAGATATTTGAACT ACA

AAGGGAACTAGCAAAGCTTACAATGAAGGCTGGAAATTCAATTTACATTCCAGGATC AA

GATTTTTTCCGACTAAAAACAATAAAAGGATGAAAGAAATGGACCAACAAGTGAAAA GTT

CAATAAAAAGCATAATTAATAAACGAGTCGTTGCGATGAAAGCTGGAGAAGCTAGGC AT

GATGATCTTTTGGGCATACTTTTGGATTCCAATTACAAAGAAATTAAACATCATGGG AAT

AGCAATTTCGGATTAAGCATCGAAGACATTATTGAAGAATGTAAACTTTTCTACTTT GCA

GGACAAGAGACCACTGGAAATATGCTTGTTTGGACTATGATTTTGTTAGGCCAACAC TC

AGAATGGCAGACACGTGCTAGAGAGGAAGTTTTGCATGTATTTGAAGATAAAAAACC AA

ATATTGATGGCTTAAGTCATCTAAAAGTGATTAGCATTATCTTTAATGAGGTTCTTA GACT

ATATTCACCAGCTGCATTTCTTAGACGCCAAATACACGAGGAAACCAAATTAGGTAA CTT

GATATTACCAGCAGGAACCCTCATCCAAATAAACGCGTTAATATTGCATCATGACAA AG

AAATGTGGGGTGAAGATGCAAATGATTTTAAACCTGAAAGATTTTATGAAGGTGTTT CAA

AGGTTACTAAAGGACAAGCTGTGTACCTTCCCTTTGGTGGCGGCCCACGTATATGCA TT

GGACAAAATTTTGCACTACTAGAAGCGAAAATGGCTCTTGCAATGATTCTACAACGC TTT

TCATTTGATTTATCGCCTTCGTACTCGCATTCTCCGCATGCTTTACCTACTCTACAA CCA

CAATTTGGTGCTCACTTGATTTTACACAAACTCTAA

Also contemplated by embodiments of the present invention is a nucleic acid encoding a polypeptide exhibiting ent-kaurenoic acid 13-hydroxylase activity and comprising a nucleotide sequence 60%, 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the sequence set forth in identity with SEQ ID NO:1 .

To determine whether a nucleic acid exhibits identity with the sequences presented herein, oligonucleotide alignment algorithms may be used, for example, but not limited to a BLAST (GenBank, see: ncbi.nlm.nih.gov/cgi-bin/BLAST/, using default parameters: Program: blastn; Database: nr; Expect 10; filter: default; Alignment: pairwise; Query genetic Codes: Standard (1 )), BLAST2 (EMBL see: embl-heidelberg.de/Services/index.html, using default parameters: Matrix BLOSUM62; Filter: default, echofilter: on, Expect: 10, cutoff: default; Strand: both; Descriptions: 50, Alignments: 50), or FASTA, search, using default parameters. Similar algorithms may be employed to determine sequence identity between two or more amino acid sequences.

In certain embodiments, there is provided a nucleic acid comprising a sequence which hybridizes to SEQ ID NO:1 , a fragment of SEQ ID NO:1 or its complement under stringent hybridization conditions. In some embodiments, the nucleic acid molecule encodes a polypeptide exhibiting ent-kaurenoic acid 13-hydroxylase activity. A worker skilled in the art would readily appreciate what is encompassed by the term "stringent hybridization conditions" (see Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982) p 387 to 389; Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1 , Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3)). Non-limiting examples of stringent hybridization conditions include hybridization in 4XSSC at 65 °C, for 8-16 hours, followed by washing in 0.1 XSSC at 65 °C for an hour, or hybridization in 5XSSC and 50% formamide at 42 °C for 8-16 hours, followed by washing in about 0.5XSSC to about 0.2XSSC at 65 °C for about 1 hour. Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). Generally, but not wishing to be limiting, stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.

Also contemplated by embodiments of the present invention are nucleic acid molecules encoding fusion proteins that exhibit ent-kaurenoic acid 13-hydroxylase activity. In some embodiments, the nucleic acid molecules encode a fusion protein comprising the sequence of SEQ ID NO:2, a variant or fragment thereof and a heterologous amino acid sequence selected from the group consisting of a membrane targeting sequence, an organelle targeting sequence, a secretion signal sequence, a purification sequence or any combination thereof.

In some embodiments, the nucleic acid molecule encodes a polypeptide that exhibits about the same activity as the polypeptide defined by SEQ ID NO:2 when tested under substantially identical conditions. However, it is also contemplated that the polypeptide may exhibit more or less activity than SEQ ID NO:2 for example, but not limited to about 50%, 60%, 70%, 80%, 90%, 100%, 1 10%, 120%, 150% or more when tested under substantially identical conditions. In some embodiments, the activity is greater than about 50%, 60%, 70%, 80%, 90%, or more. It is also contemplated that the ent-kaurenoic acid 13-hydroxylase activity of the polypeptide may be defined by an amount between the range of any two of the values listed above.

A variety of methods and assays may be employed to measure the conversion of ent- kaurenoic acid to steviol, for example, but not limited to one or more chromatographic techniques including, but not limited to high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and the like, mass spectroscopy including, but not limited to electrospray ionization (ESI), collision induced dissociation (CID) and the like or any combination thereof, for example, but not limited to liquid chromatography-electrospray mass spectroscopy (LC-ES/MS). A representative example of an assay for ent-kaurenoic acid 13- hydroxylase activity, which is not meant to be limiting is described in the examples.

In other embodiments, there is provided nucleic acids that comprise at least 10 consecutive nucleotides of SEQ ID NO:1 , for example at least 10, 12, 15, 17, 20, 21 , 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500 or more consecutive nucleotides of SEQ ID NO:1 . In an

embodiment, the nucleic acid is labeled with a marker, for example, but not limited to a fluorescent group or radioactive label that facilitates identification of the nucleotide sequence. In such a manner, the nucleic acid may be employed as a probe to detect similar or identical sequences, for example, in cells, organisms, assays or any combination thereof.

The nucleic acids described above also may be used as primers, for example, in PCR amplification reactions or the like. For example, the nucleic acids may be employed to obtain homologs of the ent-kaurenoic acid 13-hydroxylase gene. Without wishing to be limiting, this may be accomplished by contacting the DNA of a steviol-producing organism with primers under stringent hybridization conditions to permit the primers to hybridize to a ent-kaurenoic acid 13-hydroxylase gene of the organism. This may be followed by amplifying, isolating and optionally characterizing the ent-kaurenoic acid 13-hydroxylase gene from the organism.

Polypeptides

According to an embodiment of the present invention, there is provided polypeptides having ent-kaurenoic acid 13-hydroxylase activity. In an embodiment of the present invention, there is provided a polypeptide comprising an amino acid sequence defined by SEQ ID NO:2 or a fragment or variant thereof. In certain embodiments, the fragment or variant of SEQ ID NO:2 has ent-kaurenoic acid hydroxylase activity.

In certain embodiments, there is provided a polypeptide having ent-kaurenoic acid 13- hydroxylase activity and comprising about 52% to 100% sequence identity, or any amount therebetween, with SEQ ID NO: 2, for example, 52%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% identity.

In certain embodiments, there is provided a polypeptide having ent-kaurenoic acid 13- hydroxylase activity and encoded by a nucleic acid comprising a sequence having 70% 80%, 82%, 85%, 87%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% sequence identity with SEQ ID NO:1 or a sequence the hybridizes to the complement of SEQ ID NO:1 .

Also encompassed by embodiments of the present invention are polypeptides comprising a fusion protein of SEQ ID NO: 2, or a biologically active fragment or variant thereof and a heterologous amino acid sequence. Non-limiting examples of heterologous amino acid sequences include but are not limited to a membrane targeting sequence, an organelle targeting sequence, a secretion signal, an amino acid sequence that facilitates purification of the fusion protein, or a combination thereof. In an embodiment, which is not meant to be limiting in any manner, the heterologous amino acid sequence may comprise a P450 reductase, for example, but not limited to a P450 reductase from Stevia or Arabidopsis.

In some embodiments, the polypeptide exhibits about the same activity as the polypeptide defined by SEQ ID NO:2 when tested under substantially identical conditions. However, it is also contemplated that the polypeptide may exhibit more or less activity than SEQ ID NO:2 for example, but not limited to about 50%, 60%, 70%, 80%, 90%, 100%, 1 10%, 120%, 150% or more when tested under substantially identical conditions. It is also contemplated that the ent-kaurenoic acid 13-hydroxylase activity of the polypeptide may be defined by an amount between the range of any two of the values listed above.

Constructs and Hosts

Constructs

The present invention also provides constructs comprising the nucleic acids as defined above. In some embodiments, the construct is an expression vector. Appropriate expression vectors which may be used in the construction of an expression vector would be apparent to a worker skilled in the art. For example, but not limited to a recombinant expression vector, plasmid, viral vectors, artificial chromosome or the like. A worker skilled in the art could readily select appropriate expression vector for a specific host used in the methods of the invention.

Such a nucleic acid construct may comprise a variety of sequences including, but not limited to selectable marker genes, one or more origins of replication, multi-cloning or restriction endonuclease sites, and regulatory sequences including, without limitation one or more promoters, enhancers or a combination thereof.

A regulatory sequence may also include, but is not limited to promoter elements, basal (core) promoter elements, elements that are inducible in response to an external stimulus, elements that mediate promoter activity such as negative regulatory sequences or transcriptional enhancers. Regulatory sequences may also comprise elements that are active following transcription, for example, regulatory sequences that modulate gene expression such as translational and transcriptional enhancers, translational and

transcriptional repressors, upstream activating sequences, and mRNA instability

determinants. Several of these latter elements may be located proximal to the coding region. In the context of this disclosure, the regulatory sequence typically refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site.

However, it is to be understood that other nucleotide sequences, located within introns, or 3' of the sequence may also contribute to the regulation of expression of a coding region of interest. An example of a regulatory sequence that provides for the recognition for RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter sequence. A promoter sequence comprises a basal promoter sequence, responsible for the initiation of transcription, as well as other regulatory sequences (as listed above) that modify gene expression. A worker skilled in the art could readily select appropriate promoter sequences. There are also several types of regulatory sequences, including those that are

developmental^ regulated, inducible and constitutive. A regulatory sequence that is developmental^ regulated, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during the development of that organ or tissue. However, some regulatory sequences that are developmental^ regulated may preferentially be active within certain organs or tissues at specific developmental stages, they may also be active in a developmental^ regulated manner, or at a basal level in other organs or tissues within the organism as well or activated at a specific stage in an organism's life cycle.

An inducible regulatory sequence is one that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically the protein factor that binds specifically to an inducible sequence to activate transcription may be present in an inactive form which is then directly or indirectly converted to the active form by the inducer. However, the protein factor may also be absent. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus.

In respect of a cell in culture or a microorganism containing an inducible sequence exposure to an inducer may be by addition of the inducer to the culture medium. In respect of a plant, a plant containing a cell containing an inducible sequence may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods. Inducible elements may be derived from either plant or non-plant genes (e.g. Gatz, C. and Lenk, I. R. P., 1998, Trends Plant Sci. 3, 352-358).

Examples, of potential inducible promoters include, but are not limited to, Isopropyl β-D-l - thiogalactopyranoside (IPTG), tetracycline-inducible promoter (Gatz, C, 1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 89-108), steroid inducible promoter (Aoyama, T. and Chua, N. H., 1997, Plant J. 2, 397-404) and ethanol-inducible promoter (Salter, M. G., et al, 1998, Plant Journal 16, 127-132; Caddick, M. X., et al, 1998, Nature Biotech. 16, 177-180), cytokinin inducible IB6 and CKI1 genes (Brandstatter, I. and Kieber, J. J., 1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274, 982-985) and the auxin inducible element, DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971 ). A plant constitutive sequence directs the expression of a gene throughout the various parts of a plant and continuously throughout plant development. Examples of known constitutive sequences include promoters associated with the CaMV 35S transcript (Odell et al., 1985, Nature, 313: 810-812), the rice actin 1 (Zhang et al, 1991 , Plant Cell, 3: 1 155-1 165) and triosephosphate isomerase 1 (Xu et al, 1994, Plant Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejo et al, 1993, Plant Mol. Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al, 1995, Plant Mol. Biol. 29: 637-646), and the tobacco translational initiation factor 4A gene (Mandel et al, 1995 Plant Mol. Biol. 29: 995-1004). The term "constitutive" as used herein does not necessarily indicate that a gene under control of the constitutive sequence is expressed at the same level in all cell types, but that the gene is expressed in a wide range of cell types even though variation in abundance is often observed.

The nucleic acids or constructs of the present invention may be introduced into cells using any suitable transformation system known in the art. For example, but not wishing to be limiting, the nucleic acids of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro-injection, electroporation, etc. A further method for the introduction of nucleotide sequences or constructs into a plant cell is to dip developing floral tissues into a solution containing Agrobacterium tumefaciens harbouring the nucleotide construct, sucrose and a surfactant (Clough and Bent. 1998. Plant J. 16:735-743). In addition to the methods described above, several methods are known in the art for transferring DNA vectors into plant species, including gymnosperms, angiosperms, monocots and dicots (e.g. Newell. 2000. Mol.

Biotech. 16:53-65). Representative examples include DNA uptake by protoplasts, polyethylene glycol mediated uptake by protoplasts, and bombardment of cells with DNA laden microprojectiles (Plant Gene Transfer and Expression Protocols. Humana Press, Totowa, N.J.). Minor modifications to these protocols make them applicable to a broad range of plant species. For reviews of several techniques see for example Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421 - 463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. D T. Dennis, D H Turpin, D D Lefebrve, D B Layzell (eds), Addison Wesly, Langmans Ltd. London, pp. 561 - 579 (1997). Techniques for introducing the nucleic acids of the invention into bacterial, yeast or fungal cells are also known in the art.

Without wishing to be limiting, cells that comprise a nucleic acid of the present invention may be selected for by one or more selection steps. By the term "selecting" it is meant identifying cells, tissues or plants which comprise the nucleic acid of the present invention from similar cells, tissues or plants which lack the nucleic acid. Selecting may involve, but is not limited to altering the growth or development of cells, tissue or plants which lack the nucleic acid in a manner which permits such cells, tissue or plants to be differentiated or identified from plants expressing the nucleic acid of the present invention. Further, selecting may involve killing cells, tissue or plants that lack the nucleic acid. Alternatively, selecting may involve Southern hybridization to identify cells comprising the nucleic acid, Northern hybridization to identify cells comprising and expressing the nucleic acid, or Western analysis to identify cells comprising and expressing a protein, or fragment of the protein of interest, for example, an ent-kaurenoic acid 13-hydroxylase. Further, selecting may involve an enzymatic assay to measure conversion of ent-kaurenoic acid to steviol. Other selection strategies are also possible and are fully contemplated by the method of the present invention.

Hosts

The nucleic acids of the present invention may be expressed in a variety of hosts. Thus, the host may serve as an expression system for the production of ent-kaurenoic acid 13- hydroxylase.

The hosts include both prokaryotic hosts and eukaryotic hosts. For example, the host may be a bacteria, yeast, fungi, plant cells, insect cells, mammalian cells and multi-cellular organisms such as plants. Examples of prokaryotic hosts include but are not limited to Escherichia coli; Corynebacterium, Pseudomons fluorescens, Bacillus and Rhodobacter (including but not limited to Rhodobacter sphaeroides). Examples of eukaryotic systems include but are not limited to Saccharomyces cerevisiae, Shizosaccharomyces, Candida boidinii, Hansenula polymorpha, Kluyveromyces lactis, Physcomitrella, Pichia pastoris, Agaricus, Gibberella, Phanerochaete, Arxula adenivorans, Yarrowia lipolytica, filamentous fungi (including but not limited to Aspergillus, Trichoderma and Myceliophthora thermophila), insect cells, mammalian cells, plant cells and multi-cellular organisms including but not limited to plants.

In some embodiments, the host is a microorganism. In certain embodiments, the microorganism is a bacterium. In other embodiments, the microorganism is an

ascomycetes.

In some embodiments, plants and cell lines derived from such plants are used to practice the invention. Non-limiting examples of such plants include tobacco, rice, maize, Arabidopis, members of the Cruciferae family, for example, but not limited to Thalapsi arvense, members of the genus Stevia, for example, but not limited to Stevia rebaudiana, or other plants such as, but not limited to sunflower that are enriched in ent-kaurenoic acid. Accordingly, in certain embodiments, a plant cell is genetically engineered and expresses ent-kaurenoic acid 13-hydroxylase. In another embodiment, a seed, plant or plant tissue, for example, but not limited to leaves, stem, petals or the like is genetically engineered and expresses the nucleic acid of the present invention.

As an example that is not meant to be limiting in any manner, the DNA encoding ent- kaurenoic acid 13-hydroxylase can be placed under the control of the 35S enhancer- promoter plus AMV leader sequence, which may optimize transcription and translation (Kay et al. 1987. Science 236:1299-1301 ; Jobling and Gehrke. 1987. Nature 325:622-625). Other promoters such as the tCUP constitutive promoter system from tobacco can also be used to direct expression (Foster et al. 1999. Plant Mol. Biol. 41 :45-55). The nos terminator may be used to ensure stability of the resulting RNA and to terminate transcription. The completed expression vector may be cloned into a binary plasmid containing the T-DNA border sequences. This plasmid can then be transformed into Agrobacterium tumefaciens, and into a plant genome using Agrobacterium mediated transformation (Horsch et al. 1985. Science 227:1229-1231 ). The plant selectable marker can be an antibiotic such as gentamycin, hygromycin, kanamycin, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (beta-glucuronidase), or

luminescence, such as luciferase neomycin phosphotransferase may also be used.

Transformed plantlets may be selected through the selectable marker by growing the transformed cells on a medium containing the selection agent (e.g. kanamycin) and appropriate amounts of phytohormones such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.

In some embodiments, expression of the nucleic acids in non-plant cells, for example insect cells, mammalian cells, microorganisms including bacteria, yeast or fungi is contemplated. As a representative example, which is not meant to be limiting in any manner, prokaryotic organisms such as bacteria can be used to practice this invention. The DNA, or cDNA may be modified to increase translation of the mRNA encoding the polypeptide of the present invention in the desired host organism. For example, methods known in the art can be used to introduce a Ncol site at the terminus of the nucleotide sequence. A membrane anchor functional in bacteria may also be introduced. In embodiments wherein ent-kaurenoic acid hydroxylase is expressed in bacteria, a suitable P450 reductase may also be coexpressed. Further, a fusion can be created between ent- kaurenoic acid 13-hydroxylase and suitable P450 reductase, for example, but not limited to, a Stevia rebaudiana P450 reductase (Irmler et al. 2000. Plant J. 24:797-804). The fusion protein can be expressed in suitable host cells such as E. coli, for example, but not limited to BL21 , BL21 (DE3), or BL21 (DE3)pLysS, however other strains of E. coli and many other species or genera of prokaryotes may be used. As a representative example, cDNA sequences of ent-kaurenoic acid 13-hydroxylase may be transferred to expression vectors such as the commercially available pET30a, b or c (Novagen). The ent-kaurenoic acid 13- hydroxylase-P450 reductase fusion may be ligated into the pET30a plasmid. In this case, the ent-kaurenoic acid 13-hydroxylase-P450 reductase fusion would be under the control of the T7 polymerase promoter. Following induction of expression with IPTG the ent-kaurenoic acid 13-hydroxylase-P450 reductase protein may be produced, and potentially comprise up to about 50% of the total cell protein. The membrane fraction of the cells could then be isolated or whole cells lysed and used in enzyme assays, for example, for the synthesis of steviol.

As a further representative example, which is not meant to be limiting in any manner, eukaryotic microbes such as yeasts or fungi also may be used to practice this invention, including but not limited to Saccharomyces cerevisiae, although other strains and species may be used. The plasmid pYEDP60 is commonly used as an expression vector in yeast (Pompon et al. 1996. Methods Enzymol. 272:51 -64). This plasmid contains the URA3 marker that provides a selection for a mutant strain of yeast that cannot grow without uracil, such as strains WAT1 1 U and WAT21 U (Urban et al. 1997. J. Biol. Chem. 272:19176- 19186). The presence of the ura3 mutation in the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of uracil. The yeast strain WAT1 1 whose microsomal P450 reductase allele has been mutated and replaced with the Arabidopsis thaliana P450 reductase isoform 1 is a suitable for use with the pYEDP60 plasmid. When grown on media containing galactose the WAT1 1 yeast strain can over produce Arabidopsis P450 reductase isoform 1 . Other strains of yeast such as WAT21 whose microsomal P450 reductase allele has been mutated and replaced with the Arabidopsis thaliana P450 reductase isoform 2 are also suitable for use with the pYEDP60 plasmid.

It is also contemplated that ent-kaurenoic acid 13-hydroxylase- P450 reductase fusions may be expressed using the pYES/NT plasmid and a yeast strain such as, but not limited to INVSc. Following induction of expression, the membrane fraction of the cells may be isolated or whole cells lysed and used in enzyme assays, for example, for the synthesis of steviol. As will be apparent to those skilled in the art, other yeast strains and expression vectors can be used to produce polypeptides having ent-kaurenoic acid 13-hydroxylase activity. Yeast expression vectors usually have a bacterial origin of replication, a yeast origin of replication, selectable marker genes for selection of transformed cells, one or more yeast expression promoters, and a multi-cloning site for insertion of heterologous DNA sequences. Examples of other expression vectors include, but are not limited to, pESC (Stratagene), and yeast strains like G1315.

Methods

Metabolic pathways for the production of steviol and the conversion of steviol to various steviol glycosides are shown in FIG. 1 and further described in Brandle et al., (2002) Plant Molecular Biology 50: 613-622; Richman et al., (1999) The Plant Journal 19(4), 41 1 -421 ; Richman et al., (2005) The Plant Journal 41 , 55-67, which are herein incorporated by reference).

The nucleic acids of the present invention may be used in the production, isolation, purification or any combination thereof of ent-kaurenoic acid 13-hydroxylase. Further, the nucleic acids, polypeptides, cells and plants of the present invention may be used in the production, isolation, purification or any combination thereof of steviol, the primary enzyme product of ent-kaurenoic acid 13-hydroxylase, or steviol glycosides such as, but not limited to stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside, rubusoside, steviolmonoside, steviolbioside, 19-Ο-β glucopyranosol-steviol, or any combination thereof.

In an embodiment of the present invention, there is provided a method of producing steviol in a host that endogenously produces ent-kaurenoic acid comprising genetically engineering the host with a nucleic acid encoding ent-kaurenoic acid 13-hydroxylase, and expressing ent-kaurenoic acid 13-hydroxylase in the host. In this manner ent-kaurenoic acid 13- hydroxylase can convert kaurenoic acid present in the host to steviol. The steviol so produced may then be extracted from the host or, alternatively, in hosts which secrete steviol or steviol glucosides, in the culture media in which the host is maintained.

In some embodiments, the host is a microorganism. Non-limiting examples of

microorganisms include bacteria, fungi and yeast. In some embodiments, the host is a plant or plant cell, insect cell or mammalian cell. In certain embodiments, the host is a plant or a plant cell from tobacco, Arabidopis, or members of the Cruciferae family, for example, but not limited to Thalapsi arvense, members of the genus Stevia, for example, but not limited to Stevia rebaudiana, or other plants such as, but not limited to sunflower that are enriched in ent-kaurenoic acid.

In another embodiment of the present invention, there is provided a method of producing steviol in a host that endogenously produces ent-kaurenoic acid. In this method, a host is provided that comprises a nucleic acid encoding ent-kaurenoic acid 13-hydroxylase. The nucleic acid is expressed in the cell, so that the ent-kaurenoic acid 13-hydroxylase converts any ent-kaurenoic acid present in the cell to steviol. The steviol so produced may then be extracted from the host or, alternatively, in hosts which secrete steviol or steviol glucosides, in the culture media in which the host is maintained. In some embodiments, the host is a microorganism. Non-limiting examples of microorganisms include bacteria, fungi and yeast. In some embodiments, the host is a plant or plant cell, insect cell or mammalian cell.

It is also contemplated that steviol can be produced in a host, for example but not limited to, a plant, plant cell, microorganism (including but not limited to bacteria, yeast or fungi), that does not naturally produce ent-kaurenoic acid hydroxylase. Accordingly, there is provided a method of producing steviol in a host comprising genetically engineering the host with one or more nucleic acids encoding one or more enzymes that produce ent-kaurenoic acid and ent- kaurenoic acid 13-hydroxylase. In this manner one or more enzymes within the host may act on one or more substrates to produce ent-kaurenoic acid and subsequently ent-kaurenoic acid hydroxylase can convert the ent-kaurenoic acid to steviol. The steviol so produced may then be extracted from the host.

Steviol can also be produced in a host, for example but not limited to, a plant, plant cell, microorganism (including but not limited to bacteria, yeast or fungi) that does not naturally produce ent-kaurenoic acid hydroxylase, by providing a host that comprises one or more nucleic acids encoding one or more enzymes that produce ent-kaurenoic acid, and ent- kaurenoic acid 13-hydroxylase, and co-expressing the one or more nucleic acids in the host. The one or more nucleic acids may be introduced into the host via genetic engineering, or one host comprising the one or more nucleic acids, may be crossed with a second host comprising one or more other nucleic acids.

In a further embodiment, there is also contemplated downregulating the activity of ent- kaurenoic acid oxidase in a host cell, plant or enzyme system where the down regulation of oxidase activity increases the availability of kaurenoic acid for conversion to steviol through the reaction mediated by ent-kaurenoic acid 13-hydroxylase. The ent-kaurenoic acid oxidase activity may be downregulated by any suitable method known in the art, for example, but not limited to antisense, RNAi, or short-interfering RNA technology, production of dominant negatives or molecular decoys, gene knockout, and the like.

Therefore, the present invention also provides a method for steviol biosynthesis that involves providing a host that comprises a first nucleic acid encoding a sequence that down-regulates ent-kaurenoic acid oxidase expression, and a second nucleic acid that encodes ent- kaurenoic acid 13-hydroxylase, and co-expressing the first and second nucleic acids in the host. The first and the second nucleic acids may be introduced into the host via genetic engineering, or by crossing one host comprising the first nucleic acid, with a second host comprising the second nucleic acid. In certain embodiments the host is a plant or plant cell.

In a further embodiment, there is provided a method of producing one or more steviol glycosides in a host, comprising, a) selecting a host that produces ent-kaurenoic acid; b) genetically engineering the host with a first nucleic acid encoding a polypeptide having ent- kaurenoic acid 13-hydroxylase activity, and at least one second nucleic acid encoding one or more glucosyltransferases to catalyze the addition of one or more glucose molecules to steviol or a glucosylated steviol substrate, and; c) expressing the polypeptide having ent- kaurenoic acid 13-hydroxylase activity and said one or more glucosyltransferases in the host to convert ent-kaurenoic acid to one or more steviol glycosides. In certain embodiments, the host is a plant or plant cell. In other embodiments, the host is a microorganism (including but not limited to a bacteria, yeast or fungi).

The first and the second nucleic acids may be introduced into the host via genetic engineering, or by crossing one host comprising the first nucleic acid, with a second host comprising the second nucleic acid. The host may naturally produce ent-kaurenoic acid or be genetically engineered to produce ent-kauenoic acid.

In an embodiment of the present invention, which is not meant to be considered limiting in any manner, the one or more glucosyltransferases may comprise any glucosyltranferase or combination of glucosyltranferases known in the art, for example, but not limited to

UGT76G1 , UGT85C2, UGT74G1 (Richman et al. 2005. Plant J. 41 :56-67, which is incorporated herein by reference), any of the glucosyltranferases described in JP 3-277275 (which is incorporated herein by reference), or any combination thereof.

It is known that plants such as, but not limited to Arabidopsis and tobacco have the inherent ability to glucosylate steviol at the C-19 position of the C-4 carboxyl, due to native glucosyltranferase activity. Introduction of kaurenoic acid 13-hydroxylase into a host cell or plant such as, but not limited to tobacco or Arabidopsis results in the synthesis of steviol from the ubiquitous substrate kaurenoic acid, and as a result of endogenous

glucosyltranferase activity, may result in the production of 19-Ο-β glucopyranosol-steviol. Subsequent introduction of the glucosyltransferase, UGT85C2 (Richman et al. 2004. Plant J. 41 :56-67), into the host cell can catalyze the addition of glucose to the C-13 hydroxyl of 19- Ο-β glucopyranosol-steviol resulting in the production of 13,19-Ο-β glucopyranosol-steviol, also known as rubusoside.

In the absence of a native enzyme in the host cell that is able to glucosylate steviol at the C- 19 position of the C-4 carboxyl, a gene coding for the glucosyltransferase enzyme, for example, but not limited to UGT74G1 from Stevia, can be introduced to allow the synthesis of sweet steviol glycosides from kaurenoic acid. In sunflower, rubusoside could be synthesized by introducing kaurenoic acid 13-hydroxylase to allow steviol synthesis, UGT85C2 to allow steviolmonoside synthesis and UGT74G1 to allow rubusoside synthesis. Any glucosyltransferase known in the art that can glucosylate steviol resulting in any steviol glycoside may be employed herein.

In other embodiments, there is provided an in vitro method of producing steviol or one or more steviol glycosides comprising, a) reacting ent-kaurenoic acid with a polypeptide having ent-kaurenoic acid 13-hydroxylase activity under conditions to produce steviol, and; b) optionally reacting said steviol with one or more glucosyltransferases under conditions to produce one or more steviol glycosides. A worker skilled in the art would readily appreciate that the polypeptide having ent-kaurenoic acid 13-hydroxylase activity and/or the one or more glucosyltransferases may be isolated from one or more hosts which naturally express the enzymes or have been genetically engineered to express one or more of the enzymes. A worker skilled in the art would further appreciate that the enzymes may be part of a crude extract or isolated. In certain embodiments, the host is a plant or plant cell. In certain other embodiments, the host is a microorganism (including but not limited to bacteria, yeast or fungi).

Accordingly, in certain embodiments, there is provided methods to genetically engineer a host to over express polypeptide having ent-kaurenoic acid 13-hydroxylase activity and optionally the one or more glucosyltransferases. In certain other embodiments, there is provided methods to produce polypeptide having ent- kaurenoic acid 13-hydroxylase activity. In certain embodiments the method comprises a) providing a host comprising the nucleic acid of the invention and b) expressing said nucleic acid in the cell and thereby producing said polypeptide having enf-kaurenoic acid 13- hydroxylase activity.

The present invention will be further illustrated in the following examples. EXAMPLES cDNA Cloning into Plant and Yeast Expression Vectors

The nucleic acid of the invention was inserted into the pYed60 vector (Pompon) for expression in yeast, or the pCaMterX vector (Menassa, R.; Nguyen, V,; Jevnikar, A M,; Brandle, J E; (2001 ) Molecular Breeding 8:177-185) for expression in plants. One microliter of each ligation reaction was used for transforming E. coli strain XL1 -Blue MRF' cells (Stratagene). Positive colonies were identified using a PCR screening method with gene specific and vector specific primers.

cDNA Expression in Yeast

Yeast Transformation

The pYeD60 constructs were transformed into the Wat1 1 and Wat21 yeast cell lines

(Pompon). A single colony of yeast from each strain was picked using a sterile loop from a YPGA plate (20 g/l glucose, 10 g/l yeast extract, 10 g/l bactopeptone, 30 mg/l adenine, 20 g/l agar) and was used to inoculate 20 ml of liquid SC-U medium (1 .7 g/l yeast nitrogen base, 5 g/l ammonium sulfate, 0.77 g/l complete supplement mixture (amino acids) without uracil, 20 g/l glucose). The culture was grown 48 h at 30 C and 225 rpm until an OD600 of 3.2 was reached. Six millimeters of culture was added to 44 ml of YPGA liquid media (20 g/l glucose, 10 g/l yeast extract, 10 g/l bactopeptone, 30 mg/l adenine) to make 50 ml of culture at an OD600 of 0.4. This was grown for 3 h at 30EC and 225 rpm.

The cells were washed by pelleting them at 1500Xg for 15 min at room temperature and resuspending them in 40 ml of 1 XTE (10 mM Tris (pH 7.5), 1 mM EDTA

(ethylenediaminetetraacetic acid)). The cells were pelleted at 1500Xg for 15 min at room temperature, resuspended in 2 ml of 1 .times. LiAc/0.5. times. TE (100 mM LiAc (pH 7.5), 5 mM Tris (pH 7.5), 0.5 mM EDTA) and incubated at room temperature for 10 minutes.

For each transformation 1 ς of plasmid DNA and 100 ς of denatured sheared salmon sperm DNA was mixed with 100 μΙ of the yeast suspension from above 700 μΙ of

1 XLiAc/40% PEG-3350/1 XTE (100 mM LiAc (pH 7.5), 400 g/l PEG-3350 (polyethylene glycol), 5 mM Tris (pH 7.5), 0.5 mM EDTA) was added, mixed and the tubes were incubated at 30EC for 30 minutes 88 μΙ of DMSO (dimethyl sulfoxide) was added, mixed and the transformations were heat shocked at 42 C for 7 minutes. The transformations are centrifuged in a microcentrifuge for 10 s and the supernatant is removed. The cell pellets were resuspended in 1 XTE and re-pelleted. Finally the cell pellets were resuspended in 100 μΙ of 1 XTE and 50 μΙ was plated on to SC-U (1 .7 g/l yeast nitrogen base, 5 g/l ammonium sulfate, 0.77 g/l complete supplement mixture (amino acids) without uracil, 20 μΙ glucose, 20 g/l agar) selective plates and grown for 3 days at 30EC. Several colonies from each transformation picked and placed in 50 μΙ of SC-U liquid media. PCR analyses were performed to identify which ones contained the plasmid. A single colony was chosen for each construct and used in subsequent experiments.

Yeast Expression

Yeast containing the nucleic acid were streaked on to selective plates (SC-U) and grown 48 h at 30EC. A single colony from each plate was picked using a sterile loop and added to 3 ml of SC-U and grown 30 h at 30 C and 225 rpm. One milliliter of each culture was used to inoculate 25 ml of SC-U and grown for 24 h at 30 C and 225 rpm. The culture was pelleted by centrif ugation at 1500Xg and resuspended in 5 ml of YPI (medium) which was added to 20 ml of YPI (10 g/l yeast extract, 10 g/l bactopeptone, 20 g/l galactose) medium and grown for 16 h at 30EC and 225 rpm.

Ent-Kaurenoic Acid 13-Hydroxylase Analysis

A 5 ml of sample of the yeast cells which have been transformed with the nucleic acid of the invention was pelleted and resuspended in 1 ml of kaurenoic acid assay buffer (100 mM Tris (pH 7.5), 1 mM DTT (dithiothreotol), 0.5 mM NADPH (nicotinamide adenine dinucleotide phosphate), 0.5 mM FAD (flavin-adenine dinucleotide), 0.05 mg/ml kaurenoic acid,

0.05. times. Complete EDTA-free protease inhibitor cocktail (Roche), 0.4 μΜ PMSF

(phenylmethylsulphonylfluoride)) and was incubated at 30 °C for 5 h at 850 rpm in an Eppendorf Thermomixer (Westbury, N.Y.).

The culture was pelleted and the supernatant was collected and analysed by reverse-phase chromatography (C18) and negative ion ESI-MS for the presence of steviol. The analysis was done isocratically with 90:10 methanol-water. Steviol was eluted at ca. 4.5 min. Yeast cells transformed with the nucleic acid of the invention were shown to be able to convert kaurenoic acid to steviol.

cDNA Expression in Plants

Constructs generated in the pCaMterX vector were transformed into Agrobacterium tumafaciens (strain LBA4404) via electroporation. Colonies were screened by PCR to determine which ones contained the construct. Bacteria from each strain were streaked onto LB plates containing 50 μQ/m \ rifampicin, 30 μQ/m\ streptomycin, and 50 μQ/m\ kanamycin and then grown for 3 days at 28 C. A 3 ml LB culture with antibiotics was inoculated with a single colony and grown overnight at 28C and 225 rpm. One milliliter of overnight culture was used to inoculate 100 ml of LB with 50 μQ/m\ kanamycin and grown until an OD600 between 0.5 and 1 .2 was reached (approximately 18-24 h).

The culture was pelleted at 3000 g for 15 min at 4C then resuspended in freshly made 5% sucrose solution to an OD600 of 0.8. Silwet L-77 (Lehle Seeds) was added to a final concentration of 0.02% v/v. The resuspended culture was poured into a shallow container and the above ground parts of 47 day old Arabidopsis plants were submerged and agitated gently for 3 seconds. Plants were placed under a clear plastic cover for 24 h and the transformation was repeated 7 days later.

Seeds were collected upon maturity and approximately 1000 of each construct are plated on 1 /2 MS with 50 g/ml kanamycin to screen for transformants. After 14 days seedlings appearing green were transferred to soil and grown to maturity (designated as T1 plants). Leaf tissue was collected and DNA isolated to check for the presence of the transgene by PCR. Plants identified as containing the transgene were grown to maturity and seed were collected (designated T2 seed). From ten plants of each construct approximately 200 T2 seed were plated on 1 /2 MS with 50 μQ/m\ kanamycin to determine transgene copy number. Seed exhibiting a 3 to 1 ratio of kanamycin resistant to susceptible were determined to be single copy. From these plates 10 plantlets were transferred to soil and grown to maturity. Seed collected from these plants were analyzed to determine which lines were homozygous. All citations are hereby incorporated by reference.

The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.