AU779721B2 - Codeinone reductase from alkaloid poppy - Google Patents

Description

WO 00/58333 PCT/AUOO/00249 "CODEINONE REDUCTASE FROM ALKALOID POPPY" Technical Field The present invention relates to codeinone reductase from alkaloid poppy plants, the polynucleotides encoding the enzyme and to production of alkaloids from transformed poppy plants.

Background The search for useful drugs of defined structure from plants began with the isolation of morphine from dried latex, or opium, of the opium poppy Papaver somniferum in 1806 (Sertimner). The narcotic analgesic morphine and the antitussive to and narcotic analgesic codeine, the antitussive and apoptosis inducer noscapine (Ye et al., 1998), and the vasodilator papaverine are currently the most important physiologically active alkaloids from opium poppy. Of these four alkaloids, only papaverine is prepared by total chemical synthesis for commercial purposes. Opium poppy, therefore, serves as one of the most important renewable resources for pharmaceutical alkaloids. Per annum, 90-95% of the approximately 160 tons of morphine that are purified are chemically methylated to codeine, which is then used either directly or is further converted to a variety of derivatives such as dihydrocodeinone and 14-hydroxydihydrocodeinone that find use as antitussives and analgesics (Kutchan. 1998). The illicit production of morphine for acetylation to heroin is unfortunately almost ten times that amount, more than 1200 tons per year (Zenk, 1994).

The enzymatic synthesis of morphine in opium poppy has been almost completely elucidated by M.H. Zenk and coworkers and is summarized by Kutchan (1998). Opium poppy produces more than 100 different alkaloids that are derived from the amino acid L-tyrosine and have the tetrahydrobenzylisoquinoline alkaloid, (S)-reticuline. as a common intermediate. There are three NADPH-dependent reductases involved in the conversion of(S)-reticuline to morphine. (S)-Reticuline must first be converted to (R)-reticuline before the phenanthrene ring with the correct stereochemistry at C-13 can be formed. The inversion of stereochemistry at C- of (S)-reticuline occurs by oxidation to the 1.2-dehydroreticulinium ion followed by stereospecific reduction to the R-epimer by 1.2-dehydroreticulinium ion reductase [EC 1.5.1.27] (De-Eknamkul and WO 00/58333 PCT/AU00/00249 -2- Zenk, 1992). The second reduction occurs after formation of the phenanthrine nucleus with stereospecific reduction of salutaridine to salutaridinol by salutaridine reductase [EC 1.11.248] (Gerardy and Zenk, 1993). The third reduction is the penultimate step in the biosynthetic pathway to morphine, the reduction of codeinone to codeine by codeinone reductase [EC 1.1.1.2471 (Figure 1; Lenz and Zenk, 1995a,b). The substrate for codeinone reductase, codeinone, exists in an equilibrium with its positional isomer neopinone. In vitro, as codeinone is reduced, this equilibrium is continually driven from neopinone towards codeinone until the substrates are depleted (Gollwitzer et al., 1993).

Each of the known enzymes of morphine biosynthesis has been detected in both P.

somniferum plants and cell suspension culture, yet plant cell cultures have never been shown to accumulate morphine (Kutchan, 1998). Sequences of genes encoding cytochrome P450 reductases have been published in PCT/AU98/000705 which is hereby incorporated by reference.

To date, no other genes specific to morphine biosynthesis in opium poppy have been isolated. Tyrosine/dopa decarboxylase has been investigated at the molecular genetic level, but is involved in multiple biochemical processes in this plant (Facchini and De Luca, 1994). Morphine, along with the chemotherapeutic agents vincristine, vinblastine and camptothecin, is one of the most important alkaloids commercially isolated from medicinal plants. Isolation of the genes of morphine biosynthesis would facilitate metabolic engineering of opium poppy to produce plants with specific patterns of alkaloids and could ultimately lead to an understanding of the inability of plant cell cultures to accumulate morphine.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Summary of the Invention The narcotic analgesic morphine is the major alkaloid of the opium poppy Papaver somniferum. Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent. Along the morphine biosynthetic pathway in opium poppy, codeinone reductase catalyzes the NADPH-dependent reduction of codeinone to codeine. At least 10 codeinone reductase alleles are present in the genome of the poppy Papaver somniferum. Isolation, characterization and functional expression of four of the 02 9258 6999 Blake Dawson Waldron 12:16:13 16-12-2004 5 t29 112583995 3.

genes encoding codeinone reductase as described herewith enables methods for controlling alkaloid production in opium poppy plants and cultures by providing a target for genetic manipulation.

Thus, according to a first aspect, there is provided an isolated and purified polynucleotide encoding a codeinone reductase enzyme of an alkaloid poppy plant, or a variant, or analogue, or fragment thereof, having codeinone reductase activity. The polynucleotide may be selected from the group consisting of genomic DNA (gDNA), cDNA, or synthetic DNA. Preferred polynudeotides are selected from the polynucleotide sequences shown in FIGS: 10 to 15; the polynucleotide sequences which hybridize under stringent conditions to the complementary sequences of and (c) polynucleotide sequences which are degenerate to polynucleotide sequences of or It will be understood however that the sequences may be expressed in the absence of the native leader sequences or any of the 5' or 3' untranslated regions of the polynucleotide.

Such regions of the polynucleotide may be replaced with exogenous control/regulatory sequences in order to optimise/enhance expression of the sequence in an expression system.

The preferred alkaloid-producing poppy plant is Papaversomniferum.

It will also be understood that analogues and variants of the polynucleotide encoding a codeinone reductase from alkaloid poppy plants fall within the scope of the 20 present invention. Such variants will still encode an enzyme with codeinone reductase properties and may include codon substitutions or modifications which do not alter the amino acid encoded by the codon but which enable efficient expression of the polynucleotide encoding codeinone reductase enzyme in a chosen expression system.

Other variants may be naturally occurring, for example allelic variants or isoforms.

S 25 According to a second aspect there is provided an isolated and purified polynucleotide which codes for prokaryotic or eukaryotic expression of a codeinone reductase enzyme of an alkaloid poppy plant, or a variant or analogue, or fragment thereof, having codeinone reductase activity, in an environment selected from the group consisting of the extracellular environment, an intracellular membranous compartment, 30 intracellular cytoplasmic compartment or combinations thereof.

The polynucleotide encoding a codeinone reductase may be coupled to another nucleotide sequence which would assist in directing the expression of the reductase with respect to a particular cellular compartment of the extracellular environment.

According to a third aspect there is provided an isolated and purified polynucleotide which hybridises under cellular conditions with a polypeptide according to the first aspect such that expression of the polynucleotide is down regulated.

COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 02 9258 6999 Blake Dawson Waldron 12:16:43 16-12-2004 6/29 112583995 4.

Such complementary polynucleotides are useful in the present invention as probes and primers, as antisense agents or may be used in the design of other suppressive agents such as ribozymes and the like.

According to a fourth aspect there is provided a recombinant DNA construct comprising the polynucleotide according to any one of the first to third aspects.

Preferably the recombinant DNA construct is a viral or plasmid vector. Such a vector may direct prokaryotic or eukaryotic expression of the polynudeotide encoding a codeinone reductase or it may prevent or reduce its expression. The vector may also be selected from pCAl-c, pGEM-T or pFastBacl. Preferably the promoter used to control expression of the codeinone reductase gene is selected from nos, cauliflower mosaic virus or subterranean clover mosaic virus.

According to a fifth aspect there is provided an isolated and purified codeinone reductase enzyme, or a variant, or fragment thereof, being a product of prokaryotic or eukaryotic expression of the polynucleotide of the first or second aspect.

The codeinone reductase may be expressed in and by a variety of eukaryotic and prokaryotic cells and organisms, including bacteria, yeasts, insect cells, mammalian and other vertebrate cells, or plant cells. Preferably the expression system is a plant expression system and even more preferred is an alkaloid poppy plant. A suitable alkaloid poppy plant is Papaver somniferum.

20 Variants of the codeinone reductase enzyme which incorporate amino acid deletions, substitutions, additions or combinations thereof, are also contemplated. The variants can be advantageously prepared by introducing appropriate codon mutations, deletions, insertions or combinations thereof, into the polynudeotide encoding the codeinone reductase enzyme. Such variants will retain the properties of the codeinone reductase enzyme, either in vivoor in vitro, and may have improved properties. Other variants may be naturally occurring, for example allelic variants or isoforms.

As indicated above, for expression of codeinone reductase activity, a fragment of the polynucleotide encoding a codeinone reductase may be employed, such fragment encoding functionally relevant regions, motifs or domains of the reductase protein.

30 Similarly, fragments of the codeinone reductase enzyme resulting from the recombinant expression of the polynucleotide may be used. Functionally important domains of codeinone reductase may be represented by individual exons or may be identified as being highly conserved regions of the protein molecule. Those part of the codeinone reductase which are not highly conserved may have important functional properties in a particular expression system.

COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 02 9258 6999 Blake Dawson Waldron 12:17:16 16-12-2004 7 /29 112583995 According to a sixth aspect there is provided a cell transformed or transfected with a polynucleotide according to any one of the first to third aspects or a DNA construct according to the fourth aspect.

Cells which may be transfected or transformed with a polynucleotide encoding a codeinone reductase are bacteria, yeast, animal or plant cells. For preference the cells are plant cells. Even more preferred are cells from an alkaloid poppy plant, such as Papaver somniferum.

According to a seventh aspect, there is provided a callus transformed or transfected with a polynucleotide according to any one of the first to third aspects or a DNA construct according to the fourth aspect.

According to an eighth aspect, there is provided a plant transformed or transfected with a polynucleotide according to any one of the first or third aspects or a DNA construct according to the fourth aspect wherein the plant thereby exhibits altered expression of the codeinone reductase enzyme or altered codeinone reductase activity. For preference, the altered expression manifests itself in overexpression of the codeinone reductase enzyme.

However, reduced expression of codeinone reductase can also be achieved if the plant is transformed or transfected with a polynucleotide which is complementary to the polynucleotide encoding the reductase.

.Even more preferably, the transformed or transfected plant is an alkaloid poppy plant, wherein the plant has a higher or different alkaloid content when compared to a plant which has not been so transformed or transfected.

Preferably the transformed or transfected plants having higher or different alkaloid content are Papaver somniferum.

According to a ninth aspect, there is provided a method of preparing a plant which overexpresses a codeinone reductase enzyme or has altered codeinone reductase activity, *comprising transfecting or transforming a plant cell, a plant part or a plant, with the polynucleotide according to any one of the first to third aspects or a DNA construct according to the fourth aspect.

Preferably the plant overexpressing codeinone reductase is an alkaloid poppy plant and more preferably the poppy plant is Papaversomniferum. Suitable promoters to control the expression of the codeinone reductase gene may be derived from for example nos, cauliflower mosaic virus or subterranean clover mosaic virus. Other virus promoters may also be suitable. Further, the use of the endogenous promoter may also be appropriate in certain circumstances. Such a promoter may be co-isolated with the gDNA encoding the codeinone reductase enzyme.

According to a tenth aspect, there is provided a method of altering the type or blend of alkaloid in a plant comprising transforming or transfecting a plant cell, a plant COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 02 9258 6999 Blake Dawson Waldron 12:17:49 16-12-2004 8 /29 112583995 6.

part or a plant with a polynucleotide according to the first to second aspects and the polynucleotide is expressed.

According to an eleventh aspect, there is provided a method of increasing the yield of alkaloid in a plant comprising transforming or transfecting a plant cell, a plant part or plant with a polynucleotide encoding a codeinone reductase enzyme of an alkaloid producing poppy plant or a variant or fragment thereof, having codeinone reductase activity and the polynucleotide is expressed.

According to a twelfth aspect, there is provided a method of altering the type or blend of alkaloid in a plant comprising transforming or transfecting a plant cell, a plant part or a plant with a polynucleotide which hybridises under cellular conditions with a polynucleotide encoding a codeinone reductase enzyme or codes for a nucleotide sequence which hybridises under cellular conditions with the polynuceotide encoding the enzyme, such that expression of the enzyme is down regulated.

According to a thirteenth aspect, there is provided a stand of stably reproducing alkaloid poppies transformed or transfected with a polynucleotide according to any one of the first to third aspects or a DNA construct according to the fourth aspect wherein the poppies thereby exhibit altered codeinone reductase expression or altered codeinone reductase activity.

r According to a fourteenth aspect there is provided a stand of stably reproducing 20 alkaloid poppies transformed or transfected with a polynucleotide according to any one of the first to third aspects or a DNA construct according to the fourth aspect, having a higher or different alkaloid content when compared to a corresponding poppy plant of the same type which has not been so transformed or transfected.

Preferably the stably reproducing alkaloid poppy is Papaversomniferun.

According to the fifteenth aspect there is provided straw of reproducing poppies according to the fourteenth aspect having a higher or different alkaloid content when compared to straw obtained from a corresponding alkaloid producing poppy of the same type which has not been so transformed or transfected.

According to a sixteenth aspect, there is provided latex isolated from stably S 30 reproducing poppies according o the fourteenth aspect having higher or different alkaloid content when compared to latex of a corresponding alkaloid producing poppy of the same type which has not been so transformed or transfected.

According to a seventeenth aspect, there is provide an alkaloid or alkaloid concentrate isolated from straw or latex according to the fifteenth or sixteenth aspects.

Preferably the alkaloid or alkaloid concentrate comprises or incorporates one or more alkaloids selected from the group consisting of morphine, codeine, oripavine and thebaine.

COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 02 9258 6999 Blake Dawson Waldron 12:18:22 16-12-2004 9 /29 112583995 7.

According to an eighteenth aspect, there is provided a method for providing a poppy plant alkaloid or alkaloid concentrate, comprising the steps of: harvesting capsules of an alkaloid producing poppy plant transformed or transfected with a polynucleotide according to any one of the first to third aspects or a DNA construct according to the fourth aspect to produce straw; and chemically extracting one or more alkaloids from the straw; wherein the polynucleotide is expressed or effects down regulation of codeinone reductase expression in the plant.

According to a nineteenth aspect, there is provided a method for providing a poppy plant alkaloid or alkaloid concentrate, comprising the steps of: collecting and drying the latex of the immature capsules of an alkaloid poppy plant transformed or transfected with a polynudeotide according to any one of the first to third aspects or a DNA construct according to the fourth aspect to produce opium; and chemically extracting one or more alkaloids from the opium; wherein the polynucleotide is expressed or effects down regulation of codeinone reductase expression in the plant.

While it will be understood the alkaloid or alkaloid concentrate will preferably comprise or incorporate morphine, codeine, oripavine or thebaine, other intermediates in 20 the alkaloid metabolic pathway are also within the scope of the present invention, as are other mixtures of alkaloids.

i According to a twentieth aspect, the invention provides the polynucleotide sequence encoding codeinone reductase comprised in microbial deposit DSM 12737.

According to a twenty first aspect, the invention provides the polynucleotide sequence encoding codeinone reductase comprised in microbial deposit DSM 12738.

According to a twenty second aspect, the invention provides the polynucleotide S. sequence encoding codeinone reductase comprised in microbial deposit DSM 12739.

According to a twenty third aspect, the invention provides the polynuceotide sequence encoding codeinone reductase comprised in microbial deposit DSM 12740.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed anywhere before the priority date of each claim of this application.

COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 02 9258 6999 Blake Dawson Waldron 12:18:53 16.12-2004 10/29 1 12583995 8.

Unless the context dlearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense or "including, but not limited to".

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Biosynthetic pathway leading from S-Reticuline to morphine in the opium p opp y, Papa ver sonmniferum.

The reduction of codeinone to codeine by codeinone reductase drives the nonenzymatic equilibrium between neopinone and codeinone in a physiologically forward direction. The demethylation of thebaine and codeine are each thought to be catalyzed by cytochrome P450-dependent enzymes.

Figure 2. Partial amino acid sequences of native codeinone reductase.

COMS ID No: SBMW-1042515 Received by IP Australia: Time 12:33 Date 2004-12-16 WO 00/58333 PCT/AU00/00249 -9- Codeinone reductase was purified to apparent electrophoretic homogeneity from cell suspension cultures of opium poppy and hydrolyzed with endoproteinase Lys-C.

The resultant peptide mixture was resolved by HPLC and the amino acid sequences of seven peptides were obtained.

Figure 3. Amino acid sequence homology of codeinone reductase internal peptides.

Codeinone reductase peptides 3, 7, 14, 16, and 17 aligned with the reductase subunit of the 6'-deoxychalcone synthase complex from alfalfa, glycyrrhiza and soybean allowing the relative positioning of these internal peptides from opium poppy.

Figure 4. Amino acid sequence comparison of codeinone reductase isoforms.

The amino acid sequences derived from translation of the nucleotides sequences of corl.1-1.4 as compared to the reductase subunit of the 6'-deoxychalcone synthase complex from soybean (6 'dcs) indicate the very high sequence identity between isoforms (95-96%) and this reductase of phenylpropanoid metabolism The complete amino acid sequence of corl. is shown, but only those non-identical residues of the four subsequent sequences.

Figure 5. Genomic DNA gel blot analysis of the codeinone reductase gene family in opium poppy.

Genomic DNA isolated from opium poppy cell suspension cultures was hybridized to corl. I full-length cDNA and was visualized by phosphorimaging. The numbers following the restriction enzyme names indicate the number of recognition sites that occur within the corl. reading frame. This high stringency Southern analysis indicates the presence of at least ten alleles in the opium poppy genome.

Figure 6. RNA gel blot analysis of distribution of codeinone reductase transcript in a mature opium poppy.

The gel blot was prepared from RNA isolated from leaf mid rib, lateral root and 12 cm of stem tissue directly beneath the receptacle of an opium poppy plant 2 days after petal fall. 50 pg of total RNA were loaded per gel lane. The RNA was hybridized to corl.1 full length cDNA and was visualized by phosphorimagery.

Figure 7. SDS-PAGE analysis of fractions from the purification of codeinone reductase fusion protein from E. coli.

WO 00/58333 PCT/AU00/00249 Codeinone reductase was expressed as a C-terminal fusion with a 25 amino acid calmodulin-binding peptide in E. coli BL21 (DE3)pLysS. Protein bands were visualized with coomassie brilliant blue R-250. Lane 1, 15 jg crude protein from an extract of E.

coli BL21 (DE3)pLysS containing the codeinone reductase cDNA before IPTG induction; lane 2, 10 pg crude protein from an extract ofE. coli BL21 (DE3)pLysS containing the codeinone reductase cDNA 3 h after IPTG induction; lane 3, 5 Pg protein from the calmodulin affinity chromatography eluate after concentration using a Centriprep 30 column (Amicon); lane 4, Rainbow Marker protein standards (Amersham). Arrow indicates position of codeinone reductase fusion protein.

Figure 8. Chemical structures of alkaloids serving as substrates for codeinone reductase.

Of the twenty-six potential substrates tested, only seven were transformed by codeinone reductase. The names of the untransformed compounds are given in the Description of Preferred Embodiments. Codeinone is the physiological substrate for this enzyme in most, if not all. varieties of opium poppy. Morphinone also serves as a physiological substrate in Tasmanian varieties. The Km values provided for those seven substrates were determined for COR1.3.

Figure 9. Proposed alternative biosynthetic pathway leading from thebaine to morphine in opium poppies from Tasmania.

This alternative biosynthetic pathway was proposed after oripavine was discovered in Tasmanian varieties of opium poppy (Brochmann-Hanssen, 1984).

Codeinone reductase from non-Tasmanian varieties can also catalyze the reduction of morphinone to morphine (Lenz and Zenk, 1995b). COR .1 -COR1.4 each catalyzed this reduction with equivalent specific activity. The demethylation of thebaine and codeine are thought to be catalyzed by cytochrome P450-dependent enzymes.

Figure 10. cDNA sequence of corl.1.

Figure 11. cDNA sequence of corl.2.

Figure 12. cDNA sequence of corl.3.

Figure 13. cDNA sequence of corl.4.

Figure 14. Partial cDNA sequence of Figure 15. Partial cDNA sequence of corl.6.

WO 00/58333 PCT/AU00/00249 11 Description of the Preferred Embodiments cDNas that encode codeinone reductase were isolated. Four full-length reading frames and two partial clones (FIGS 10 to 15) were isolated that represent six alleles from a gene family that may have at least 10 members. An analysis of RNA and enzyme activity from various stages of developing opium poppy seedlings and roots, stem, leaf and capsule of mature poppy plants indicated that transcript from these alleles is present throughout the plant at all developmental stages, with the highest total enzyme activity being in the capsule after petal fall. This would suggest that morphine biosynthesis occurs in all major plant organs starting within the first seven days after seed germination. Biosynthesis of morphine continues throughout the life cycle of this annual with the highest biosynthetic activity taking place in the capsule after petal fall, consistent with the amount of biosynthetic enzyme present. The amount of extractable RNA remained high in the capsule until three days after petal fall, after which time the quantity of extractable RNA decreased rapidly.

A biochemical analysis of four functionally expressed alleles, corl.1-corl. 4, revealed no significant differences in the temperature or pH optima, Km values or substrate specificity of the isoforms. All isoforms were able to reduce morphinone to morphine.

Purification and Amino Acid Sequence Analysis of Opium Poppy Codeinone Reductase Codeinone reductase was purified to apparent electrophoretic homogeneity from opium poppy cell suspension cultures and the amino acid sequence of seven endoproteinase Lys-C-generated peptides was determined (Figure A comparison of these amino acid sequences with those available in the GenBank/EMBL sequence database allowed a relative positioning of peptides 7, 14 and 16 due to sequence homology with an NADPH-dependent reductase from members of the Fabaceae alfalfa, glycyrrhiza and soybean (6'-deoxychalcone synthase) that synthesizes 4,2',4'-trihydroxychalcone in co-action with chalcone synthase (Figure 3) (Welle et al., 1991). PCR primers were then designed based on the codeinone reductase peptide sequences. The sequences of the primers used in the first round of PCR were: WO 00/58333 PCT/AU00/00249 -12- CTT TTT ATA ACT TCT AA-3' (derived from Peptide 14) and G C C C G C

T

3'-GTG GTC TAA CGT CAI CGT TCI CCT TT-5' (derived from Peptide 7) A AG C Resolution of an aliquot of the first PCR experiment by agarose gel electrophoresis revealed a mixture of DNA products, none of which was the expected band of approximately 480 bp. This was presumably due to the relatively low specificity of the degenerate primers coupled to a low abundance of codeinone reductase transcript.

Another aliquot of the first PCR reaction mixture was, therefore, used as template for nested PCR with the following primers: CTT TTT ATA ACT TCT AA-3' (same as Peptide 14 primer above) and G C C C G C

T

3'-CAI CAC TTA GTT CAC CTT TAC-5' (nested primer derived from Peptide 16) G C C to yield an approximately 360 bp DNA fragment and the following primers to yield an approximately 180 bp DNA product: GTI AAC CAA GTI GAA ATG AGI CCI AC-3' (nested primer derived from T G G TC Peptide 16) and 3'-GTG GTC TAA CGT CAI CGT TCI CCT TT-5' (same as Peptide 7 primer above) A AG C The results from the nested PCR were bands of the expected size. The translation of the nucleotide sequences of these PCR products indicated that they encode codeinone reductase.

Isolation of cDNAs Encoding Codeinone Reductase Screening of approximately 200,000 clones of a primary cDNA library prepared from opium poppy RNA isolated from capsule and cell suspension culture did not result in the identification ofcodeinone reductase clones. Likewise, difficulty was also confronted with detecting a band on RNA gel blots that corresponds to the size expected for codeinone reductase. In order to overcome the apparent problem of low steady state levels of codeinone reductase transcript, RACE-PCR was used to generate both the WO 00/58333 PCT/AU00/00249 13and 3'-portions of the cDNA (Frohman, 1993). A series of non-degenerate primers based on the nucleotide sequence information determined for the PCR product generated as described in the previous section were used for and 3'-RACE. The nucleotide sequence of the resultant and partial clones were thus determined in three major fragments and suggested the presence of isoforms. The full length cDNA clones were then generated by RT-PCR using the following primers and RNA isolated from opium poppy cell suspension culture as template: '-ATG GAG AGT AAT GGT GTA CCT-3' (located at the 5'-terminus) and 3 '-TCT ACC ATT CAC TCC TGA CAG-5' (located in the 3'-flanking region) followed by nested PCR with the following primer pair: GCT AGC ATG GAG AGT AAT GGT GTA CCT ATG-3' (located at the Nhe 1 5'-terminus) and 3'-CTT CTC AAG ACC CTA CTC TTC CTA CCT AGG GAA-5' (located at the Ban HI 3'-terminus).

The PCR product was digested with the restriction endonucleases Nhe I Barn HI, ligated into Nhe I Bam HI digested pCAL-c and transformed into Escherichia coli BL21(DE)pLysS. Each cDNA was hence constructed in frame in front of DNA encoding a 25 amino acid long calmodulin-binding peptide to facilitate eventual heterologous protein purification. Single colonies were grown in 3 ml medium and were assayed for the ability to reduce codeinone. Of forty colonies tested, ten were found to contain functional enzyme. Nucleotide sequence determination of these ten cDNAs resulted in the identification of four alleles encoding codeinone reductase. The analogous PCR products had also been prepared with the cDNAs placed behind the calmodulin-binding peptide gene in pCAL-n-EK, but only the C-terminal fusion proteins bound the calmodulin affinity resin, indicating that the amino terminus of the fusion protein lies within the folded polypeptide.

By sequence comparison, codeinone reductase clearly belongs to the aldo/keto reductase family, a group of structurally and functionally related NADPH-dependent oxidoreductases. Members of this family possess three consensus sequences that are also positionally conserved: aldo/keto reductase consensus 1 (amino terminus) G WO 00/58333 PCT/AU00/00249 -14- X X X X X EXX G [corl.1 G Y R H F D T A A A Y Q T E E C L aldo/keto reductase consensus 2 (central) X X X X X X X X X G N (F,Y) [corl. I M E E C Q T L G F T R A I G V C N aldo/keto reductase consensus 3 (carboxy terminus) X X X R X X (G,S,T,A,E,Q,K) X (L,I,V,M,FA) [corl. V V K S F N E A R M K E N L K This third consensus sequence is centred around a lysine residue, the modification of which has been shown to affect the catalytic efficiency of aldose and aldehyde reductases (Morjana et al., 1989).

The four functional full-length cDNAs (corl. 1, corl.2, corl.3 and corl.4) encoding codeinone reductase share approximately 95-96% sequence identity (Figure 4).

These sequences are comprised in microbial deposit Nos. DSM 12737, DSM 12738, DSM 12739 and DSM 12740 respectively, deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) of Mascheroder Weg Ib, D-38124 Braunschweig, Germany on 16 March 1999. In addition, a similar cDNA generated by PCR (cor2) was 70% identical to the codeinone reductase cDNAs, but was not functional. These opium poppy cDNAs were 53% identical to soybean NADPH-dependent reductase 6'-deoxychalcone synthase (Welle et al., 1991) (Figure 4), 33% identical to rat 3-hydroxysteroid dehydrogenase [EC 1.1.1.50], 38% identical to bovine prostaglandin F synthase [EC 1.1.1.188], 37% identical to apple D-sorbitol-6-phosphate dehydrogenase [EC1.1.1.200], 38% identical to bacterial (Pseudomonas putida) morphine 6-dehydrogenase [EC 1.1.1.218] and 35% identical to yeast (Pichia stipitis) xylose reductase (Amore at al., 1991).

Genomic DNA Analysis and Gene Expression Pattern Genomic DNA was used as template for a PCR analysis of corl. 1-corl.4. Each gene was found to contain one intron that was conserved in size (443 bp) and location (beginning after nucleotide +561) within the open reading frame, but not in nucleotide sequence. In comparison. cor2 contained two introns beginning after nucleotides +321 and +514. Genomic DNA gel blot analysis using corl. 1 as hybridization probe resulted in a complex hybridization pattern that suggests the presence of at least ten genes that could encode codeinone reductase in opium poppy (Figure From the isolation and WO 00/58333 PCT/AU00/00249 nucleotide sequence analysis of cDNA clones, it is certain that at least six of these ten genes are expressed in the plant and plant cell suspension culture. (Two additional partial cDNAs (corl.5 and corl.6; FIGS 14 and 15) were generated by RT-PCR using plant RNA as template.) When the peptide sequences presented in Figure 2 are compared with the translations of the cDNA sequences in Figure 4, it is clear that a mixture of isoforms was purified for amino acid sequence analysis. From the initial biochemical analysis of codeinone reductase, evidence for only two isoforms in the poppy plant and one isoform in poppy cell suspension culture was observed (Lenz and Zenk,1995b).

RNA gel blot analysis indicated the presence of a very weakly hybridizing RNA of approximately 1.4 kb in poppy leaf, root and stem of a mature plant two days after petal fall (Figure Since corl transcript was apparently present at very low levels, further analysis was undertaken by nested RT-PCR. Morphinan alkaloids begin to accumulate rapidly in poppy seedlings four to seven days after germination (Rush et al., 1985; Wieczorek et al., 1986). An analysis ofcodeinone reductase enzyme activity and transcript accumulation showed that enzyme activity is at 310 pkat/g dry tissue weight (dwt) already at day seven after germination (Table This activity remains at that level throughout a three week growth period, then decreases to 148 pkat/g dwt by the eighth week. In comparison, opium poppy cell suspension culture also contains 330 pkat/g dwt enzyme activity. Transcript was detected by RT-PCR for corl. -corl.4 at all developmental stages. Since two PCR amplifications were necessary in order to detect cor1 transcript, a comparative quantitation was not undertaken.

The distribution of codeinone reductase enzyme activity and transcript was also investigated in mature opium poppy plants two days after petal fall. On a dry tissue weight basis, most activity was present in the capsule (730 pkat/g dwt), then the lateral root (560 pkat/g dwt) followed by stem and leaf lamina (Table Again, no differences could be found in the distribution pattern of the four isoforms by RT-PCR.

WO 00/58333 PCT/AUOO/00249 -16- Table 1. Analysis of codeinone reductase enzyme activity and transcript in developing opium poppy and in plant suspension culture.

Plant Plant age Specific activity Total activity Transcript material (days) (pkat/mg) (pkat/dwt) detection S- 7 11 310 A 14 9 330 n S21 8 310 S-56 12 150 7 10 330 Presence of transcript in each RNA population was determined by performing two nested PCR amplifications as described in the Examples.

Substitute Sheet (Rule 26) RO/AU WO 00/58333 PCT/AUOO/00249 16a- Table 2. Analysis of codeinone reductase enzyme activity and transcript in developing opium poppy two days after petal fall.

Plant Specific activity Total activity Transcript part (pkat/mg) (pkat/dwt) detection a Capsule Stem b Leaf lamina 730 250 Lateral root 560 SPresence of transcript in each RNA population was determined by performing two nested PCR amplifications as described in the Examples.

hStem tissue beginning at the receptacle and extending 12 cm downwards was extracted. Plants were approximately 120cm high.

Substitute Sheet (Rule 26) RO/AU WO 00/58333 PCT/AU00/00249 17- Functional Characterization of the Codeinone Reductase Alleles The four codeinone reductase isoform-calmodulin-binding peptide fusion proteins were purified from E. coli lysates in one step with a calmodulin affinity column.

Beginning with 250 mg total protein in the bacterial extract, 10.5 mg codeinone reductase with a specific activity of 5.2 nkat/mg protein could be obtained in 73% yield.

Aliquots from a typical purification analyzed by SDS-PAGE are shown in Figure 7.

Codeinone reductase purified by this method is nearly homogeneous and demonstrated properties that compared favourably to those of the native enzyme (Lenz and Zenk, 1995b).

The temperature optimum, pH optimum and Kn2 values for codeinone, codeine, NADPH and NADP were determined for each of the isoforms (Km values are indicated in Table Significant differences in these values were not found. For all isoforms, the temperature optimum for reduction (physiologically forward reaction) was 28 0 C, for oxidation (physiologically reverse reaction) was 30°C, the pH optimum for reduction was 6.8 and for oxidation was 9.0. The isoforms were also tested for their ability to transform morphinan alkaloids structurally related to codeinone and codeine. The reductive reaction with NADPH as cofactor functions with morphinone, hydrocodone and hydromorphone as substrate. The oxidative reaction with NADP as cofactor functions with morphine and dihydrocodeine as substrate. The Km values for, and structures of, these additional substrates with COR1.3 are shown in Figure 8. In all cases, the physiologically forward reaction yielded lower Km values than the physiologically reverse reaction, with codeinone having the lowest Km value at 48 p M.

No differences in temperature or pH optimum were observed whether codeinone or morphinone were used as substrate in the assay. NADH could not substitute for NADPH with any of the isoforms. Tritium was enzymatically transferred to codeinone from [4R- 3 HJNADPH, but not from [4S- 3 H]NADPH. indicating that codeinone reductase stereospecifically abstracts the pro-R hydrogen from the cofactor.

WO 00/58333 PCT/AU00/00249 18- Table 3. Comparison of properties of codeinone reductase isoforms COR1.1 COR1.2 COR1.3 COR1.4 Amino acid identity 100 95 96 96 K, codeinone (pM) 58 62 48 K, NADPH (pM) 180 220 205 197 K, codeine (pM) 220 200 187 140 K, NADP (pM) 53 58 45 Calculated Mr 35,808 35,704 35,797 35,705 Calculated pi 6.25 5.71 6.32 6.33 The reduction of codeinone to codeine is the last of three NADPH-dependent reductions that occur along the biosynthetic pathway leading from (S)-reticuline to morphine in opium poppy. The two other potential substrates for reduction, the 1,2dehydroreticulinium ion and salutaridine (Figure or for the physiologically reverse reaction, salutaridinol and (R)-reticuline, were tested as substrates; with the codeinone reductase isoforms. None of these alkaloids served as substrate indicating that codeinone reductase can catalyze only one reductive step in morphine biosynthesis. In addition, the following analogs were also inactive: and (R)-norreticuline, (S)-reticuline and norcodeine.

Since codeinone reductase showed sequence similarity to several members of the aldo/keto reductase family, a series of substrates were tested to reflect members from carbohydrate and steroid metabolism. D-Sorbitol-6-phosphate, D-xylose, prostaglandin DI, 5-androstene-3p,17p-diol, 5a-androstan- 17p-ol-3-one, 5a-cholestane-3p-ol, p-estradiol, cyclohexanone and 2-cyclohexene-l-one were not transformed by codeinone reductase. The highest amino acid sequence identity was, however, to the reductase subunit of the 6'-deoxychalcone synthase complex from soybean (Welle et al., 1991). In order to test for a functional evolutionary relationship between isoflavonoid and alkaloid anabolism. codeinone reductase was analyzed for the ability to substitute for the reductase in the formation of 6-deoxychalcone in co-action with either native chalcone synthase or native stilbene synthase from Pinus sylvestris. In the presence of 4-coumaryl-CoA, malonyl-CoA, NADPH, chalcone synthase and codeinone reductase or cinnamoyl-CoA. malonyl-CoA, NADPH, stilbene synthase and codeinone reductase, WO 00/58333 PCT/AU00/00249 19formation of product was not observed. Likewise, the reductase of the 6'-deoxychalcone synthase complex could neither reduce codeinone in the presence of NADPH nor oxidize codeine in the presence of NADP.

Example 1 Purification of Native Enzyme and Amino Acid Sequence Analysis Cell suspension cultures of the opium poppy Papaver somniferum were routinely grown in either 1-litre conical flasks containing 400 ml of Linsmaier-Skoog medium (Linsmaier and Skoog, 1965) over 7 days at 23 C on a gyratory shaker (100 rpm) in diffuse light (750 lux). Differentiated opium poppy plants were grown outdoors in Upper Bavaria. Seedlings were grown on substrate from 7 to 56 days in a greenhouse at 0 C, 65% relative humidity and 12 h cycles of light and dark.

A mixture of codeinone reductase isoforms was purified from opium poppy cell suspension cultures exactly according to Lenz and Zenk (1995b). The purified enzyme preparation was subjected to SDS/PAGE to remove traces of impurities and the coomassie brilliant blue R-250-visualized band representing codeinone reductase was digested in situ with endoproteinase Lys-C as reported in (Eckerskom and Lottspeich, 1989, Dittrich and Kutchan, 1991). The peptide mixture thereby obtained was resolved by reversed phase HPLC [column, Merck Lichrospher RP18; 5 [im (4 x 125 mm); solvent system. 0.1% trifluoroacetic acid, 0.1% trifluoroacatic: acid acetonitrile; gradient of 1 per min; flow rate of Iml/min] with detection at 206 nm.

Microsequencing of seven of the peptides thus purified was accomplished with an Applied Biosystems model 470 gas-phase sequencer.

Example 2 Generation of Partial and Full-Length cDNAs from Opium Poppy Partial cDNAs encoding codeinone reductases from opium poppy were produced by PCR using cDNA produced by reverse transcription of total RNA isolated from 3 to suspension cultured cells. DNA amplification using either Taq or Pfu polymerase was performed under the following conditions: 4 min at 94 0 C, 35 cycles of 94 0 C, 30 sec: 45 0 C. 30 sec; 72 0 C. 1 min. At the end of 35 cycles, the reaction mixtures were incubated for an additional 5 min at 72 0 C prior to cooling to 4 0 C. Reamplification WO 00/58333 PCT/AU00/00249 of DNA using nested primers was performed as above, but the primer annealing temperature was raised from 45 to 55 0 C. The amplified DNA was then resolved by agarose gel electrophoresis, the bands of approximately the correct size were isolated and subcloned into pGEM-T (Promega) prior to nucleotide sequence determination. The specific sequences of the oligodeoxynucleotide primers used are indicated above.

Total RNA was isolated and RNA gels were run and blotted as previously described (Pauli and Kutchan, 1998). Genomic DNA was isolated and DNA gels were run and blotted according to Bracher and Kutchan (1992). cDNA clones were labelled by random-primed labelling with [a-32P]dCTP and oligodeoxynucleotides were end-labelled with [y- 3 2 P]ATP. Hybridized RNA on Northern blots and DNA on Southern blots were visualised with a Raytest BAS-1500 phosphorimager. The entire nucleotide sequence on both DNA strands of full-length cDNA clones in either pGEM-T or pCAL-c was determined by dideoxy cycle sequencing using internal DNA sequences for the design of deoxyoligonucleotides as sequencing primers.

The sequence information requisite to the generation of full-length cDNAs was derived from the nucleotide sequences of the partial cDNAs generated as described above. The complete nucleotide sequence of one reading frame was determined using codeinone reductase specific oligodeoxynucleotide primers in and 3'-RACE-PCR experiments with a Marathon T M cDNA amplification kit (Clontech). RACE-PCR was performed using the PCR cycles described above. The amplified DNA was then resolved by agarose gel electrophoresis and the band of the approximate expected size was isolated, subcloned into pGEM-T and sequenced.

Nested primer pairs were then used to generate full-length clones for heterologous expression by RT-PCR using opium poppy cell suspension culture RNA as template. The final primers used in clone amplification contained the restriction endonuclease recognition sites Nhe I and Bam HI that were appropriate for subcloning directly into the pCAL-c (Stratagene) expression vector. The specific sequences of these primers are indicated above. RT-PCR was carried out using the PCR cycles given above. The amplified DNA was then resolved by agarose gel electrophoresis and the band of the correct size (972 bp) was excised and isolated for further subcloning into the expression vector.

WO 00/58333 PCT/AU00/00249 -21- Example 3 Heterologous Expression and Enzyme Purification Full-length cDNAs generated by RT-PCR were ligated into p-CAL-c and transformed into the E.coli strain BL21 (DE3)pLysS. For enzyme assays, single colonies S were picked and grown in 3 ml Luria-Bertani medium containing 100 Pg/ml ampicillin at 37°C to an OD 590 of 0.8. For protein purification, single colonies were picked and grown in 1 1 Luria-Bertani medium containing 100 pg/ml ampicillin at 37°C to an OD 590 of 1.8. Cells were collected by centrifugation 5 min at 4,000 x g and 4°C. The bacterial pellet was resuspended in either 0.1 M potassium phosphate buffer pH 6.8 for the reduction of codeinone or 0.1 M glycine buffer pH 9 for the oxidation of codeine. The bacterial pellet from a 3 ml culture was resuspended in 0.5 ml buffer and that from a one litre culture in 100 ml buffer. The cells were ruptured by sonication. Cellular debris was removed by centrifugation 5 min at 4,000 x g and 4°C and the supemantant used directly for either affinity chromatography purification using the AffinityTM Protein Expression and Purification System according to the manufacturer's instructions (Stratagene) or for enzyme activity measurements according to Lenz and Zenk (1995b).

Example 4 Enzyme Assay and Product Identification The oxidative and reductive reactions catalyzed by codeinone reductase were assayed according to Lenz and Zenk (1995b). The oxidation of codeine to codeinone by heterologously expressed enzyme in a crude bacterial extract was used for large scale production of enzymic product for structure elucidation by 'H NMR, 13C NMR and mass spectrometry. The enzyme assays were extracted twice with two volumes of CHCI 3 the combined organic phase was reduced in vacuo and resolved by semipreparative HPLC using the following gradient: [column, Knauer LiChrosopher 100 RP18 endcapped; lpm (16 x 250 mm); solvent system, 97.99% (vlv) H 2 0 2% CH 3 CN, 0.01% (v/v)

H

3

PO

4 1.99% H 2 0, 98% CH 3 CN, 0.01% H 3

PO

4 gradient: 0-9 min 0-8% B, 9-24 min 8% B. 24-45 min 8-25% B, 45-75 min 25% B, 75-75.3 min 25-0% B, 75.3-90 min 0% B; flow 4.5 ml/min] with detection at 204 nm using authentic codeine (retention WO 00/58333 WO 0058333PCT/AUOO/00249 22 time, 38 min) and codeinone (retention time, 49 min) as reference materials. In this manner. 10 mg codeinone was enzymically produced and purified.

Codeinone- 1H(36OMHz. CDC1 3 1.87 (1IH,dd J15a115e12.2, 15e/16a3.1, H-15e), 2.08 (I~dd,~j5'j1'i4.5, J1 512, 5e 1n) 2.2 9 (1 H, ddd,Ji~~ 12.3, ~IeIa3.1, i 3.1,J 16 I 1.8,Hil6,). 2.35 (1H, dd,Joaii0e 18.5 ,Jg9II,5.9H-10a), 2.47 (3H,5, CH 3

N-

.6 d, 16 1 1.8Jiaiie45,H16), 3.12 (1IH, d, ~laIe18.5,H- 10), 3.21 (1IH, m, H- 14), 3.43 (1 H, m, 3.85 s, CH 3 4.71 (1 H, s, 6.09 (1 H, dd, J17/8 1, J7/14 2.8, 6.62 (1IH, d, J11 2 8.3, H-i1), 6.66 (1IH, dd, J 7 8 10. 1, 8114 1.5, H-8), 6.68 (1IH, d, J 112 8.3, 3 C(90.6 MHz, CDCI 3 20.5 3 3.8 (C-i15), 41.3 14), 42.8 (NMe), 43.0 13), 46.8 16), 56.8 (OMe), 59.1 88.0 114.8 119.9 125.7 (C-i 11), 128.9 (C-i 12), 132.6 142.6 144.9 148.7 (C- 194.4 El-MS (70 eV),nv'z 297 100%, 282 268 254 23 8 229 214 188 (15) 165 152 13 9(16), 128 115 (4 1).

Example Transformation of plants with nucleotide sequences from genes encoding codeinone reductase proteins.

Plant Materials Two plant lines were used in transformation experiments. These were Nicotiana tahacum line Wisconsin38, and Papaver somniftruni line C048. Preparation of plant materials and tissue culture and transformation conditions were as described in An et.al (1986), Hooykaas and Schilperoort (1992) and PCT Application PCT/AU99/00004, all of which are incorporated herein by reference.

Bacterial strains and vectors The disarmed Agrobacterium tumnefaciens strain LBA4404 was used in transformation experiments. DNA constructs capable of expressing the codeinone reductase genes were prepared in a binary vector containing a 35S-nptll selectable marker, and transformed into the N.iabacum and P. somniferum lines.

Successful transformation of these plant lines was achieved as judged by regeneration of X' iahacum plants on medium containing 1 00mg/I kanamycin indicating expression of the nptll selectable marker, which was verified by NPTII WO 00/58333 PCT/AU00/00249 -23enzyme assays. Coexpression of the codeinone reductase gene was determined by RT- PCR (reverse transcriptase polymerase chain reaction) assay.

successful selection of transformed cell cultures of P.somniferum using the same nptlI selectable marker indicative of expression from the vector, followed by the generation of typel and typell embroyogenic callus prior to the production of transformed plants.

Thus, the identification and cloning of genes for codeinone reductase from P.somniferum now provides a means by which alteration of the enzymatic step(s) involving this can be achieved. The overexpression of these sequences can be achieved using vectors which express one or more of the codeinone reductase alleles, while downregulation of general codeinone reductase activity or the activity of specific alleles can be achieved using vectors expressing antisense, ribozymes, plus-sense cosuppression or RNAi sequences from regions conserved between the codeinone reductase alleles or other sequences which are unique to each allele. These genes encoding the sense, antisense, ribozyme, RNAi or other such sequences can be delivered as transgenes stably integrated into the poppy genome or transiently in the form of a viral vector.

Although the invention has been described with reference to specific embodiments, modifications that are within the knowledge of those skilled in the art are also contemplated as being within the scope of the present invention.

WO 00/58333 PCT/AU00/00249 -24-

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Claims (27)

1. 28. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS 1. An isolated and purified polynucleotide encoding a codeinone reductase enzyme of an alkaloid poppy plant or a variant, or analogue, or fragment thereof, having codeinone reductase activity. 2. A polynucleotide according to claim 1, selected from the group consisting of genomic DNA, cDNA, or synthetic DNA. 3. A polynucleotide according to claim 1 or claim 2, selected from the group consisting of: the polynucleotide sequences shown in FIGS 10 to the polynucleotide sequences which hybridize under stringent conditions to the complementary sequences of and polynucleotide sequences which are degenerate to the polynucleotide sequences of or 4. A polynucleotide according to any one of the preceding claims, lacking a native 15 leader sequence or one or more 5' and/or 3' untranslated regions of the polynucleotide. 5. A polynucleotide according to claim 4, wherein the native leader sequence or one or more of the 5' and/or 3' untranslated regions are replaced with exogenous regulatory sequence which regulates, optimises or enhances expression of the 20 polynucleotide in an expression system. S 6. A polynucleotide according to any one of the preceding claims which encodes a codeinone reductase enzyme of Papaversomniferum. 7. A polynudeotide according to any one of claims 2 to 6, which is a synthetic polynucleotide comprising one or more codons preferred for expression in plant cells. 8. An isolated and purified polynucleotide which codes for prokaryotic or eukaryotic expression of a codeinone reductase enzyme of an alkaloid poppy plant, or a variant, or analogue, or fragment thereof having codeinone reductase activity, in an COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 02 9258 6999 Blake Dawson Waldron 12:19:29 16-12-2004 12/29 112583995
2. 29. environment selected from the group consisting of the extracellular environment, an intracellular membranous compartment, intracellular cytoplasmic compartment or combinations thereof. 9. A polynucleotide according to claim 8, comprising a nucleotide sequence which directs expression of the codeinone reductase enzyme with respect to a particular cellular compartment or the extracellular environment. A polynucleotide according to any one of claims 1 to 9, wherein the variant has greater than 70% amino acid sequence homology with the codeinone reductase enzyme. 11. An isolated and purified polynucleotide having a sequence which hybridises under cellular conditions with a polynucleotide according to any one of claims I to such that expression of the polynucleotide is down-regulated. 12. A recombinant DNA construct comprising the polynucleotide according to any one of claims 1 to 13. A recombinant DNA construct comprising the polynucleotide according to claim 11. 14. A DNA construct according to claim 12 or 13, which is a viral or plasmid vector. S* 15. A DNA construct according to claim 12 or 13, capable of directing prokaryotic or eukaryotic expression of the polynucleotide. 16. A DNA construct according to any one of claims 12 to 15, comprising a promoter for controlling the expression of the polynucleotide. 17. A DNA construct according to claim 16, wherein the promoter is endogenous. 18. A DNA construct according to claim 16, wherein the promoter is derived from nos, cauliflower mosaic virus or subterranean clover mosaic virus. 19. A DNA construct according to claim 12 or 13, wherein the plasmid is pCLA-c. 20. A DNA construct according to claim 12 or 13, where the plasmid is pGEM-T. 21. A DNA construct according to claim 12 or 13, where the plasmid is pFastBacl. COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 61 2 92586999 Blake Dawson Waldron 15-58:25 20-1 2-2004 3 /4 112602361 22. A method of providing a codeinone reductase enzyme which has involvement in an alkaloid biosynthesis pathway of a poppy plant, or a variant, or fragment thereof, having codeinone reductase activity, comprising the steps of: transfecting a cell with a polynucleotide encoding the enzyme as defined in any one of claims 1 to 10 or a DNA construct according to claim 12, or the variant, or fragment thereof; culturing the cell under conditions such that the polynucleotide is expressed; and purifying the enzyme, or the variant or fragment thereof. 23. A method according to claim 22 wherein the polynucleotide has a nucleotide sequence selected from the group consisting of: polynucleotides shown in FIGS 10 to 15, with or without a native leader sequence or one or more untranslated sequences of the polynucleotide; regions of encoding fragments which have codeinone reductase activity; and polynucleotide sequences which are degenerate to or 24. A method according to claim 22 or 23 wherein the variant has greater than amino acid sequence homology with the codeinone reductase enzyme. 25. A method according to any one of claims 22 to 24 wherein the cell is a plant cell. 20 26. A method according to any one of claims 22 to 25 wherein the poppy plant is Papa ver somniferum. 27. An enzyme provided by a method as defined in any one of claims 22 to 26 or the variant or fragment thereof provided by the method. 28. A cell transformed or transfected with a polynucleotide according to any one of claims 1 to 11 or a DNA construct according to any one of claims 12 to 21. 29. A cell according to claim 28, wherein the cell is a plant cell derived from an alkaloid poppy plant. A cell according to claim 29, wherein the poppy plant is Papaversomruifrum.
3. 31. A cell according to claim 28, which is a bacterial cell. COMS ID No: SBMI-01047094 Received by IP Australia: Time 16:02 Date 2004-12-20 02 9258 6999 Blake Dawson Waldron 12:20:18 16-12-2004 14 129 112583995 31.
4. 32. A cell according to claim 28, which is an animal cell.
5. 33. A cell according to claim 28, which is a yeast cell.
6. 34. A callus transformed or transfected with a polynucleotide according to any one of claims 1 to 11 or a DNA construct according to any one of claims 12 to 21.
7. 35. A plant transformed or transfected with a polynucleotide according to any one of claims 1 to 11 or a DNA construct according to any one of claims 12 to 21, wherein the plant thereby exhibits altered expression of the codeinone reductase enzyme or altered codeinone reductase activity.
8. 36. A plant according to claim 35, wherein the altered expression is overexpression of codeinone reductase enzyme.
9. 37. A plant according to claim 35, wherein the altered expression is reduced expression of the codeinone reductase enzyme.
10. 38. A plant according to any one claims 35 to 37, which is an alkaloid poppy plant.
11. 39. A plant according to claim 38, wherein the plant has a higher or different alkaloid 4 content when compared to a plant which has not been so transformed or transfected.
12. 40. A plant according to claim 38 or claim 39, wherein the alkaloid poppy plant is S* Papaver somniferum.
13. 41. A method of preparing a plant which overexpresses a codeinone reductase enzyme or has altered codeinone reductase activity, comprising transfecting or transforming a plant cell, a plant part or a plant, with the polynucleotide according to any one of Sclaims 1 to 10 or a DNA construct according to claims 12. S42. A method according to claim 41, wherein the plant is an alkaloid producing poppy plant. 25 43. A method according to claim 42, wherein the poppy plant is Papaversomniferum.
14. 44. A method of altering the type or blend of alkaloid in a plant comprising transforming or transfecting a plant cell, a plant part or a plant with a polynucleotide encoding a codeinone reductase enzyme of an alkaloid producing COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 02 9258 6999 Blake Dawson Waldron 12:20:42 16-12-2004 15129 112583995 32. poppy plant, or a variant or fragment thereof having codeinone reductase activity, wherein the polynucleotide is a polynucleotide as defined in any one of claims 1 to or a DNA construct as defined in claim 12, and is expressed. A method of increasing the yield of alkaloid in a plant comprising transforming or transfecting a plant cell, a plant part or plant with a polynuceotide encoding a codeinone reductase enzyme of an alkaloid producing poppy plant, or a variant, or fragment thereof having codeinone reductase activity, wherein the polynucleotide is a polynucleotide as defined in any one of claims 1 to 10 or a DNA construct as defined in claim 12, and is expressed.
15. 46. A method of altering the type or blend of alkaloid in a plant comprising transforming or transfecting a plant cell, a plant part or a plant with a polynucleotide which hybridises under cellular conditions with a polynucleotide encoding a codeinone reductase enzyme or codes for a nucleotide sequence which hybridises under cellular conditions with the polynucleotide encoding the enzyme, such that expression of the enzyme is down regulated.
16. 47. A stand of stably reproducing alkaloid producing poppies transformed or transfected with a polynucleotide according to any one of claims 1 to 11 or a DNA construct according to any one of claims 12 to 21, wherein the poppies thereby exhibit altered codeinone reductase expression or altered codeinone reductase 20 activity.
17. 48. A stand of stably reproducing alkaloid producing poppies according to claim 47, having a higher or different alkaloid content when compared to a corresponding poppy plant of the same type which has not been so transformed or transfected. S:I 49. A stand of stably reproducing alkaloid poppies according to claim 47 or claim 48, 25 wherein the alkaloid poppy is Papaversomniferum. Straw isolated from stably reproducing poppies according to any one of claims 47 to 49, having a higher or different alkaloid content when compared to straw obtained from a corresponding alkaloid producing poppy of the same type which •has not been so transformed or transfected.
18. 51. Latex isolated from stably reproducing poppies according to any one of claims 47 to 49, having a higher or different alkaloid content when compared to latex of a COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 02 9258 6999 Blake Dawson Waldron 12:21:10 16-12-2004 16/29 112583995 33. corresponding alkaloid producing poppy of the same type which has not been so transformed or transfected.
19. 52. An alkaloid or alkaloid concentrate isolated from the straw as defined in claim 50 or the latex as defined in claim 51.
20. 53. An alkaloid or alkaloid concentrate according to claim 52, comprising or incorporating one or more alkaloids selected from the group consisting of morphine, codeine, oripavine and thebaine.
21. 54. A method for providing a poppy plant alkaloid or alkaloid concentrate, comprising the steps of: harvesting capsules of an alkaloid producing poppy plant transformed or transfected with a polynucleotide according to any one of claims 1 to 11 or a DNA construct according to any one of claims 12 to 21 to produce straw; and chemically extracting one or more alkaloids from the straw; wherein the polynuceotide is expressed or effects down regulation of codeinone reductase expression in the plant.
22. 55. A method for providing a poppy plant alkaloid or alkaloid concentrate, comprising Sthe steps of: e collecting and drying the latex of the immature capsules of an 20 alkaloid poppy plant transformed or transfected with a polynucleotide according to Sany one of claims 1 to 11 or a DNA construct according to any one of claims 12 to 21 •to produce opium; and chemically extracting one or more alkaloids from the opium; wherein the polynucleotide is expressed or effects down regulation of codeinone reductase expression in the plant.
23. 56. A method according to claim 54 or claim 55, wherein the alkaloid or alkaloid concentrate comprises or incorporates one or more alkaloids selected from the group consisting of morphine, codeine, oripavine and thebaine.
24. 57. An alkaloid or alkaloid concentrate provided by a method as defined in any one of claims 54 to 56.
25. 58. The polynudeotide sequence encoding codeinone reductase comprised in microbial deposit DSM 12737. COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16 0292586999 Blake Dawson Waldron 02 958 699 Bake awso Wairon12:21:35 16-12-2004 1 2 17/29 112583995
26. 59. The polynudleotide sequence encoding codeinone reductase comprised in microbial deposit DSM 12738. The polynudleotide sequence encoding codeinone reductase comprised in microbial deposit DSM 12739.
27. 61. The polynucleotide sequence encoding codeinone reductase comprised in microbial deposit DSM 12740. Dated: 15 December 2004 Johnson Johnson Research Pty Limited Patent Attorneys for the Applicant: BLAKE DAWSON WALDRON PATENT SERVICES COMS ID No: SBMI-01042515 Received by IP Australia: Time 12:33 Date 2004-12-16