WO2006010246A1 - The isolation and characterization of a gene encoding a littorine mutase/hydroxylase, transgenic plants and uses thereof for alerting alkaloid biosynthesis - Google Patents

Abstract

A gene encoding a littorine mutase/hydroxylase has been isolated and characterized. The gene is used in processes of altering alkaloid biosynthesis. Transgenic plants containing the gene have been produced and used for the production of tropane alkaloid.

Description

The isolation and characterization of a gene encoding a littorine mutase/hydroxylase, transgenic plants and uses thereof for altering alkaloid biosynthesis.

CROSS REFERENCE TO RELATED APPLICATION[S] [0001] This application claims priority to U.S. Provisional Patent

Application 60/592,144, filed July 30, 2004, the content of the entirety of which is incorporated by this reference.

TECHNICAL FIELD [0002] The present invention relates generally to biotechnology. More particularly, the present invention relates to the production of transgenic plants and processes of altering alkaloid biosynthesis.

BACKGROUND OF THE INVENTION [0003] Tropane alkaloids such as scopolamine and hyoscyamine are synthesized in many solanaceous plants including Atropa, Hyoscyamus and Datura (1-4). The compounds represent an important class of plant-derived drugs with uses based on their muscarine (smooth muscle) anticholinergic properties. Atropine (racemic hyoscyamine), for example, is used as a premedication for anesthesia to prevent bronchial secretions and block bradycardia associated with some anesthetic drugs. It is also used as a mydriatic to dilate pupils prior to ophthalmological examinations.

[0004] Despite considerable efforts over many years and good progress towards elucidating the biosynthesis of tropane alkaloids in the Solanaceae, knowledge of the pathway remains incomplete (3). For example the formation of tropinone from Λ/-methylpyrrolinium remains puzzling. Another mystery is the nature of the carbon skeleton rearrangement which occurs during the conversion of (/?)-littorine to (S)-hyoscyamine (see FIG. 2). In this conversion, the 3-phenyllactate moiety of littorine is rearranged to tropate. Isotope labeling studies have indicated that C1' and C3' form the phenylacetate moiety of hyoscyamine, with C2' forming the hydroxylmethyl group in a stereochemically- controlled reaction (5-7). [0005] The question has been raised as to what types of enzymes are involved in the conversion of littorine to hyoscyamine. An enzyme with mechanism and properties similar to vitamin Bi2-dependent isomerases has been suggested (1). Retey and coworkers have provided evidence for such a littorine isomerase which requires S-adenosylmethionine as a cofactor (8). On the other hand, labeling and inhibitor studies and mechanistic considerations, led Robins and coworkers to propose that a cytochrome P450 is involved, with the formation of hyoscyamine aldehyde as an intermediate (3, 9, 10). If the latter proposal is correct, the cytochrome P450 enzyme involved would be unusual in its catalysis of a carbon skeleton rearrangement, concomitant with oxidation of the littorine substrate (11).

[0006] In addition to learning more about the conversion of littorine to hyoscyamine, it would also be of commercial benefit to produce higher levels of these tropane alkaloids in plant species as well as producing related alkaloids that are used commercially. One method of enhancing the production of tropane alkaloids may be accomplished through breeding and selection programs, as well as genetic engineering techniques using known genes in the tropane alkaloid pathway. (Jouhikainen et al. 1999).

[0007] The tropane alkaloids (-)-hyoscyamine, atropine (the racemic form of (-)-hyoscyamine) and scopolamine are esters of tropic acid and the tropane derivatives tropine or scopine. (see, FIG. 1 ). These tropane alkaloids are synthesized in the roots and other parts of various solanaceous plants. The literature on tropane alkaloid biosynthesis includes numerous revisions in an incomplete pathway for producing the tropane alkaloids, which vary from species to species, (see, Facchini 2001; Hemscheidt 2000; Humphrey & O'Hagan 2001 ; O'Hagan & Robins 1998).

[0008] Tropine is thought to be formed from ornithine or arginine via putrescine as illustrated in FIG. 1. Putrescine is methylated and pxidatively deaminated to 4-aminobutanal, which undergoes spontaneous cyclization to form the N-methyl-Δ¹-pyrrolinium cation. It is suggested that tropinone is formed by acylation of this cation with acetonedicarboxylic acid (derived from acetate) followed by an intramolecular condensation to from the azabicyclic compound tropinone (Humphrey & O'Hagan 2001 ). The gene encoding a stereospecific NADPH-dependent enzyme called tropinone reductase I that produces tropine has been cloned.

[0009] The biosynthesis of the tropic acid moiety of the tropane alkaloids has also undergone recent revision (O'Hagan & Robins 1998). Phenylalanine is oxidatively deaminated to phenylpyruvate and reduced to phenyllactate. The phenyllactate, possibly in coenzyme A thioester form, is esterfied to tropine to give littorine, a previously unsuspected intermediate of hyoscyamine and scopolamine biosynthesis. Isomerization of the ester results in the conversion of the phenyllactate moiety to tropate. Based on inhibition by clotrimazole, the latter reaction may be cytochrome P450-mediated.

[0010] Further, hyoscyamine may be converted to its epoxide, scopolamine, by hydroxylation and epoxide formation catalyzed by a cloned bifunctional enzyme, hyoscyamine 6β-hydroxylase. (Facchini 2001). To date, the enzymes for which the corresponding genes have been cloned include putrescine, N-methyltransferase, tropinone reductase and hyoscyamine 6β- hydroxylase.

SUMMARY OF THE INVENTION [0011] The present invention discloses an enzyme involved in the biosynthesis of tropane alkaloids. In one embodiment, the enzyme comprises a littorine mutase/hydroxylase. The reaction catalyzed by the enzyme and the product produced by the reaction have not been previously reported. A gene encoding the enzyme shares sequence similarity with plant cytochrome P450 genes.

[0012] In one embodiment, an isolated or recombinant nucleic acid sequence encoding a littorine mutase/hydroxylase is disclosed. The isolated or recombinant nucleic acid sequence comprises a nucleotide sequence selected from the group consisting of a DNA sequence having the sequence of SEQ ID NO: 1, a nucleic acid sequence that hybridizes to SEQ ID NO: 1 or its complementary strand, and a nucleic acid sequence that would hybridize to SEQ ID NO: 1 or its complementary strand, but for the degeneracy of the genetic code. Fragments of the isolated or recombinant nucleic acid sequence having the same function as the isolated or recombinant nucleic acid sequence of the present invention are also within the scope of the present invention.

[0013] In one embodiment, a process for growing a transgenic plant, plant seed, or progeny thereof includes planting a transgenic plant, plant seed or progeny thereof having means for modifying iittorine to form a tropane alkaloid is stably integrated therein. The transgenic plant, plant seed or progeny thereof to maturity is grown to maturity and tissue from the mature transgenic plant, plant seed or progeny thereof is harvested. To extract the tropane alkaloid, an intermediate product such as a homogenate, tincture, oil, infusion, or exudates is produced from the tissue, and the tropane alkaloid is isolated from the intermediate product. The transgenic plant, plant seed or progeny thereof may be of a Solanaceae origin. Other plants that may be transformed with the isolated or recombinant nucleic acid of the present invention include plants of a Erythroxylum origin (i.e., cacao or cocoa).

[0014] As used herein, the phrase "plant, plant seed or progeny thereof will be used to refer to a plant or its progeny or tissue. For instance, "plant, plant seed or progeny thereof will refer to the T1 , T2 and T3 generation of a plant as well as to plants produced with asexual reproduction methods. [0015] In another embodiment, the isolated or recombinant nucleic acid sequence encoding the Iittorine mutase/hydroxylase is used to genetically engineer and, thus, produce a transgenic plant, plant seed or progeny thereof that overproduces tropane alkaloids of commercial interest, such as by overcoming any biochemical limitation at the level of Iittorine (see, FIG. 1). [0016] In a further embodiment, the isolated or recombinant nucleic acid sequence encoding the Iittorine mutase/hydroxylase is inserted in a vector. The isolated or recombinant nucleic acid sequence encoding the Iittorine mutase/hydroxylase may be inserted such that regulatory sequences in the vector direct expression of isolated or recombinant nucleic acid sequence encoding the Iittorine mutase/hydroxylase. The vectors may be expression vectors or cloning vectors. For the transgene (i.e., the isolated or recombinant nucleic acid sequence encoding the Iittorine mutase/hydroxylase) to be expressible, it may be operatively coupled to a promoter in the vector. The promoter may be temporal, tissue specific or constitutive. The transgene may also be operatively coupled to a terminator sequence.

[0017] In another embodiment, the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase or a vector including the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase may also be associated with or include a marker gene that enables a transformed cell (i.e., plant or tissue culture cell) containing the transgene to be distinguished from other cells that do not include the transgene. Expression of the marker may be controlled by a promoter that allows expression in a cell in culture, thus, allowing for the selection of a cell or tissue containing the marker at any stage of regeneration of the cell or tissue.

[0018] In yet an additional embodiment, a host cell transformed with a vector having the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase is also disclosed. The host cell may comprise a microbial host cell transformed with the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase.

[0019] In a further embodiment, a vector including the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase or other DNA construct including the transgene may be introduced into a plant cell using any suitable means. Any method that provides for the stable incorporation of the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase in a plant cell or any species is within the scope of the present invention. Thus, the present invention also discloses a plant cell transformed with the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase.

[0020] In a further embodiment, a protein or peptide encoded by the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase is disclosed. Thus, the present invention further includes an isolated protein that is the expression product of the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase. The protein may be expressed in a host cell harboring the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase in an expression vector. In one embodiment, the protein encoded by the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase comprises an enzyme having littorine mutase/hydroxylase activity. In another embodiment, the protein comprises SEQ ID NO: 2.

10021] In yet another embodiment, an antibody capable of binding to a protein of the present invention is disclosed. In one embodiment, the antibody may be obtained by injecting an isolated or recombinant littorine mutase/hydroxylase protein of the present invention in a mammal, and isolating the antibodies capable of binding the isolated or recombinant littorine mutase/hydroxylase protein produced by the mammal.

[0022] In another embodiment, a modified littorine mutase/hydroxylase protein produced by known methods and having substantially the same function as the littorine mutase/hydroxylase of the present invention is disclosed. In one embodiment, the function that such modified littorine mutase/hydroxylase proteins includes is the ability to modify littorine to form a tropane alkaloid. In some embodiments, the modified littorine mutase/hydroxylase possess greater heat stability, improved kinetic characteristics, improved specificity for littorine, or other enhanced or similar function as the littorine mutase/hydroxylase of the present invention.

[0023] In a further embodiment, a plant, a plant cell or progeny thereof having a transgenic littorine mutase/hydroxylase enzyme is disclosed. The littorine mutase/hydroxylase enzyme may be encoded by the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase stably transformed into the plant, plant cell or progeny thereof. In one embodiment, the plant, plant cell or progeny thereof may be of the Solanaceae family.

[0024] In yet an additional embodiment, a process for producing a tropane alkaloid includes cultivating a transgenic plant, plant seed or progeny thereof having an isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase integrated therein. The process further includes harvesting tropane alkaloids produced by the plant, plant seed or progeny thereof trom a tissue or component of the plant, plant seed or progeny thereof. In one embodiment, the tropane alkaloid may be produced by the transformation of littorine to produce hyoscyamine, scopolamine or 3-(3- phenylglycerolyoxy)tropane. Tropane alkaloids produced by the process are also included. Other tropane alkaloids that may be produced include, without limitation, benzatropine, homatropine, novatropine, scopolamine, methscopolamine bromide, cocaine, xanthine alkaloids, or other alkaloids from various plant species.

[0025] . Use of a composition comprising a tropane alkaloid harvested from a transgenic plant, plant seed or progeny thereof of the present invention for the manufacture of a medicament for the treatment or administration of an anticholinergic drug to a subject in need thereof is further disclosed.

[0026] In a further aspect, the invention discloses the use of a composition comprising antibodies or fragments thereof that bind to an isolated or recombinant littorine mutase/hydroxylase for the manufacture of a kit for detecting the littorine mutase/hydroxylase is also disclosed.

[0027] A plant or progeny thereof transformed with the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase expresses the nucleic acid sequence in at least some of the cells of the plant or progeny thereof such as the roots of the plant or progeny thereof.

[0028] In one embodiment, a method for reducing the expression of a gene encoding a littorine mutase/hydroxylase includes the use of an RNAi agent specific for the gene encoding a littorine mutase/hydroxylase. The RNAi may be specific for the isolated or recombinant nucleic acid sequence encoding the littorine mutase/hydroxylase and used to block one or more steps of the tropane alkaloid biosynthesis pathway.

[0029] In another embodiment, a process for producing a precursor or intermediate compound of the tropane alkaloid biosynthesis pathway comprises blocking one or more steps of the tropane alkaloid biosynthesis pathway in a cultivated cell, plant, plant seed or progeny thereof. The desired compounds are harvested from cell, plant, plant seed or progeny thereof. BRIEF DESCRIPTION OF THE DRAWINGS [0030] FIG. 1 shows steps in a pathway for forming tropine. [0031] FIG. 2 illustrates late steps in the proposed tropane alkaloid biosynthetic pathway. [0032] FIG. 3 depicts effects of suppression of HnCYPI gene expression on alkaloid accumulation in Hyoscyamus. FIG. 3A₁ VIGS of HnCYPI in H. muticus; FIG. 3B, RNAi of HnCYPI in H. niger. H. niger hairy roots were generated from A. rhizogenes containing no vector (WT)₁ empty pH7GWIWG2(ll) vector (VC), or pRL041 in three separate hairy root lines (HR13, HR16 and HR19); Tissue levels are shown for littorine (gray), 3'- hydroxylittorine (black), hyoscyamine (striped) and scopolamine (white). Means and standard errors are shown.

[0033] FIG. 4 illustrates a phylogenetic analysis of H. niger littorine mutase/monooxygenase and related CYP proteins. An unrooted distance tree shows the relationship of littorine mutase/monooxygenase to various cytochrome P-450 (hereinafter referred to as "CYP") subfamilies. CYP classification, species and Genbank accession numbers are as follows: 75A1, CYP75A1 Petunia x hybrids Z22544; 75B, CYP75B4 Perilla frutescens AB045593; 76A, CYP76A2 Solanυm melogena X71657; 76B, CYP76B6 Catharanthus roseus AJ251269; 76C, CYP76C2, Arabidopsis thaliana AY062600; 76E, CYP76E1 Medicago truncatula unpublished; 76F₁ CYP76F7 Hypericum androsaemum AY596977; 76G, CYP76G1 Arabidopsis thaliana NM_115157; 76H, CYP76H4 Oryza sativa AK070050; 80A1 , CYP80A1 Berberis stolonifera U09610 ; 80B2, CYP80B2 Coptis japonica AB025030. [0034] FIG. 5. Expression of HnCYPI in wild type and modified plant tissues. RT-PCR of HnCYPI for A) various tissues of H. niger, B) H. muticus control and HnCYPI VIGS plants; C) transgenic tobacco hairy roots and D) transgenic H. niger hairy roots with HnCYPI suppressed by RNAi (D). VC, vector control. [0035] FIG. 6 shows the GC analysis of trimethylsilylated alkaloids extracted from tobacco hairy root cultures expressing HnCYPL FIG. 6A₁ Line 1 (overexpressing HnCYPI) without (/?)-littorine; FIG. 6B, Vector control line supplied with (R)-littorine; FIGS. 6C and D, Line 1 supplied with (R)-littorine. Chromatograms shown are from retention time 16.5 min to 17.1 min and are normalized to the eicosane internal standard. Chromatogram d is a 10X magnification of c. [0036] FIG. 7 depicts the conversion of tropane alkaloids by HnCYPI in yeast microsomes. Gas chromatograms of trimethylsilylated alkaloids extracted from microsomes obtained from the yeast strains WAT11/pRL039 (expressing HnCYPI ; B and D) and WAT11/pRL039 (empty vector; A and C) after incubation with (R)-littorine (A and B) or hyoscyamine aldehyde (C and D). Note the ordinates of A and B are FID response and those of C and D are single ion monitoring signal at m/z = 124. Chromatograms are normalized to the scopolamine internal standard (S). Peaks were assigned to underivatized parent compounds as follows: 1 , 3-phenylacetoxytropane; 2, homatropine; 3, hyoscyamine; 4, littorine; 5 and 6, hyoscyamine aldehyde (trans and cis isomers of the enol form); 7, 3'-hydroxylittorine; 8, 2'- hydroxyhyoscyamine; 9 and 10, unknown.

[0037] FIG. 8 shows tropane alkaloid reactions catalyzed by control and littorine monooxygenase-containing yeast microsomes. This represents a proposed interpretation of the data from assays with littorine, hyoscyamine and 3'-hydroxylittorine. Solid arrows, littorine mutase/monooxygenase in yeast microsomes; Open arrows, control yeast microsomes derived from WAT11/p YES-DEST52.

[0038] FIG. 9 depicts a mechanistic rationale for the products of HnCYPL Hydrogen abstraction at vivinal positions and oxygen rebound with optional rearrangement could give rise to three products from littorine and hyoscyamine.

[0039] FIG. 10 is a genetic map of the vector pRL042.

BEST MODE OF THE INVENTION [0040] The present invention discloses the characterization and use of a littorine mutase/hydroxylase gene and an expression product thereof. [0041] As part of a functional genomics program to study the molecular genetics of tropane alkaloid biosynthesis, transport and regulation, an investigation of the hypothesis that a cytochrome P450 was involved in littorine conversion was undertaken. The approach included the generation of expressed sequence tags from a Hyoscyamus niger L. cDNA library and the testing of candidate cytochrome P450 gene function by virus induced gene silencing (VIGS).

[0042] VIGS exploits the RNA silencing pathway directed against invading viruses to silence the expression of host genes (12). It has been used to silence genes involved in a variety of plant biosynthetic pathways (13-17). Tobacco rattle virus (TRV), a tobravirus with a bipartite ssRNA genome that silences genes in Nicotiana benthamiana (18, 19) and other Solanaceae (20), has been reported to infect H. niger (21). In related work, proof-of-concept experiments showed that TRV-mediated gene silencing was effective in H. muticus (Egyptian henbane) for phytoene desaturase and known alkaloid biosynthetic enzymes.

[0043] In the present invention, the use of VIGS was used to elucidate the function of candidate cytochrome P450 genes of H. niger for their involvement in littorine rearrangement. This led to the discovery of one such cytochrome P450 and the characterization of its unusual catalytic repertoire through heterologous expression.

[0044] The cloning of the isolated nucleic acid encoding a littorine mutase/hydroxylase (i.e., HnCYPI ) represents a successful use of EST analysis in combination with virus-induced gene silencing (hereinafter, "VIGS") for functional genomics in a non-model plant species. The VIGS, RNAi, native expression and tobacco overexpression data support the discovery of the HnCYPI as a root-specific cytochrome P450 involved in the conversion of littorine to hyoscyamine, as well as the production of other tropane alkaloids such as 2'-hydroxyhyoscyamine. As late as 1998, evidence for the role of a SAM-dependent enzyme with similarities to vitamin Bi₂-dependent enzymes was put forth (8). On the other hand, Robins, O'Hagan and coworkers have provided data and rationale favoring the involvement of a cytochrome P450 (3, 9, 41 ). The data presented here clearly support the latter view.

[0045] The yeast expression data, although complicated by endogenous yeast enzyme activities, provide important insights into the nature of littorine mutase/monooxygenase. As summarized in FIG. 8, four reactions can be attributed to the enzyme - a) littorine rearrangement/dehydrogenation, b) littorine hydroxylation, c) hyoscyamine hydroxylation and d) hyoscyamine dehydrogenation. Three of the four reactions can be rationalized to involve initial attack at the benzylic position of littorine or hyoscyamine (3, 10). The case of littorine rearrangement has been discussed at length by O'Hagan and Robins (10).

[0046] FIG. 9 illustrates the proposed cytochrome P450-catalyzed free radical mechanism for littorine rearrangement. An alternative carbocation- based mechanism is also possible (10). As for typical cytochrome P450 hydroxylation reactions (11), the free radical mechanism includes hydrogen abstraction and rebound steps, but also includes an unusual rearrangement. The resulting gem-diol then dehydrates to the aldehyde. The oxygen rebound and dehydration steps resemble the mechanism proposed for cytochrome P450-mediated alcohol dehydrogenation (42). [0047] If oxygen rebound were to occur without rearrangement, then 3'- hydroxylittorine would be formed, as observed. It is notable that the stereochemistry of the 3'-hydroxylittorine product is as expected if it shares an intermediate with the rearrangement reaction resulting from removal of the 3'- pro-R hydrogen (10)(see FIG. 9). [0048] If abstraction of a benzyl hydrogen were to occur on hyoscyamine, followed by oxygen rebound, the expected product would be 2'- hydroxyhyoscyamine, us observed. Qn the other hand, attack at C3' of hyoscyamine would give the aldehyde in a reaction which may share an intermediate with littorine rearrangement (see FIG. 9). Thus, the products of HnCYPI from two substrates can be rationalized by the involvement of two vicinal sites of initial oxidation, typical of cytochrome P450s, and one optional, but rather unique, rearrangement. [0049] The importance of the observed reactions in yeast microsomes as compared to the situation in tropane alkaloid-producing plants including Hyoscyamus is not entirely clear. The tobacco expression data supports the role of the littorine mutase/hydroxylase enzyme primarily in accumulating hyoscyamine. Thus, it appears that in plants the conversion of littorine to hyoscyamine aldehyde is an important reaction. As suggested previously, it is likely that a separate activity is involved in reduction of the aldehyde to hyoscyamine (3). On the other hand, the present invention discovered the accumulation of the alternate HnCYPI product, 3'-hydroxylittorine, indicating that hydroxylation of littorine without rearrangement also occurs in plants. Based on tests with 3'-hydroxylittorine as a substrate, it appears that this may be a dead end product, rather than an intermediate in hyoscyamine formation.

[0050] It is not likely that the observed oxidation of hyoscyamine by HnCYPI is an important reaction in plants. This may be due to competition from hyoscyamine aldehyde hydrogenation or by relatively rapid removal of the aldehyde product from the enzyme in plant tissues.

[0051] The invention will be described in more detail with reference to the following examples. The examples serve only to illustrate the invention.

EXAMPLES

Example I.

[0052] A cultured-root-minus leaf subtracted cDNA library was prepared from Hyoscyamus niger. Approximately 3,000 cDNA clones were isolated and their inserts were sequenced. BLAST analysis indicated that the insert of clone pRL033 shared sequence similarity to plane cytochrome P450 genes. In order to investigate the function of the gene corresponding to pRL033, virus-induced gene silencing (VIGS) studies were performed in Hyoscyamus muticus. The insert of pRL033 was cloned into a VIGS vector (pYL156) designed for use in tobacco. (Liu et al. 2002). [0053] The resulting plasmid and appropriate control plasmids were used separately to transform Agrobacterium tumafaciens. To effect virus- induced gene silencing, the resulting Agrobacterium tranformants were 2005/000826

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infiltrated into the leaves of separate H. muticus plants. After five weeks, the leaves of these plants were harvested. Tropane alkaloids were extracted and analyzed by gas chromatography as described in Drager 2002. Relative to control leaves, leaves from the plants transformed with the VIGS plasmid containing the pRL033 insert showed increased levels of littorine and reduced levels of hyoscyamine and scopolamine.

Example II.

[0054] To confirm the identity of the gene corresponding to pRL033, a cDNA containing the full-length open reading frame was cloned using RACE.

The DNA sequence of the full-length cDNA clones is shown in SEQ ID NO: 1.

The corresponding amino acid sequence of the open reading frame is shown in

SEQ ID NO: 2.

Example III.

[0055] The open reading frame was cloned into a yeast expression vector (pYES-DEST52). The yeast, Saccharomyces cerevisiae (WAT11 , which expresses a plant cytochrome P450 reducatase) was transformed with the resulting plasmid using uracil autotrophy for selection. [0056] After induction on galactose, yeast cells containing either empty vector or vector and pRL033 were collected by centrifugation and lysed by agitation with glass beads. Microsomes were prepared by ultracentrifugation and assayed for total protein. Samples of microsomes containing equal amounts of total protein were incubated with and without littorine and analyzed by GC-MS after trimethylsilylation. While no (expected) hyscyamine product was detected from these assays, samples containing the pRL033 gene product and littorine gave a novel GC peak with an El mass spectrum which included an apparent molecular ion of m/z 449 and a diagnostic ion of m/z 179. This peak was assigned to the di(trimethylsilyl)-derivative of 3-(3-phenylglyceroyloxy) tropane. A peak with the same retention time and mass spectrum was found upon analysis of H. niger roots. The data indicates that the gene corresponding to pRL033 is a littorine mutase/hydroxylase involved in the biosynthesis of hyoscyamine and scopolamine from littorine.

Example IV. [0057] Chemical synthesis.

[0058] (R)- and (S)-littorine enantiomers were synthesized essentially as described previously (22) from tropine (Fluka) and 3-phenyllactic acids (Aldrich) by heating over dry HCI(g).

[0059] Methyl (2/^:?,3f?)-2₎3,-dihydroxy-3-phenylpropionate was derived from ethyl (2R,3S)-3-phenylglycidate (a kind gift of B. Sharpless). Similarly, a mixture of diastereomers of methyl 2,3-dihydroxy-3-phenylpropionate was derived from ethyl 3-phenylglycidate (Aldrich).

METHODS Example V.

[0060] . Plant sample preparation.

[0061] Plant samples for alkaloid analysis were lyophilized and weighed. Each sample was homogenized in 4 ml ethanol/NH₄OH (5% (v/v) aqueous NH₄OH solution (30%) in ethanol) and sonicated in a water bath for 20 minutes. The sample was centrifuged and the supernatant was removed. The pellet was extracted twice with 2 ml aliquots of ethanol/NH₄OH. The pooled extracts were dried under N₂, taken up in 1 ml 0.1 N HCI and centrifuged. The supernatant was neutralized with 100 μl 1 N sodium carbonate (pH 10) and applied to an Extrelut QE column (1 ml capacity, EM Science Gibbstown, NJ). After 15 min, 10 ml dichloromethane was applied and the eluate collected. Eicosane (Aldrich) was added to the sample as an internal standard and the solvent was removed under a nitrogen stream. For GC or GC/MS analysis, the residue was dissolved in 250 μl of /V,O-fo/s(trimethylsilyl)acetamide (BSA; Aldrich)/pyridine (1 :1).

Example Vl.

[0062] Isolation of (2'/^:?_/3'/^:?)-3'-hydroxylittorine from H. niger roots. [0063] Two hundred grams of lyophilized H. niger roots were extracted as described above except that two successive 500 ml ethanol/NH₄OH extractions were used and three 3 ml capacity Extrelut QE columns were each eluted with 30 ml dichloromethane. A compound subsequently identified as (2'R,3'R)-3'-hydroxylittorine was purified by HPLC fractionation of the pooled eluates using an Agilent 100 series HPLC with fraction collector and eluting through a series of two 4.6 mm X 125 mm Whatman partishpere C-18 columns at 1 ml/min, starting with 50 mM phosphate buffer at pH 8.0 with a linear gradient to 50% acetonitrile in 20 min. This yielded 125 μg of chromatographically pure compound. GC/MS analysis of the trimethylsilylated compound indicated a molecular ion of m/z 449, fragment ions associated with an unmodified tropane ring (including m/z 124) and of m/z 179 consistent with 2 hydroxyl groups present on the phenylpropane moiety of the molecule. The underivatized compound shows a predictable pattern of ions consistent with the proposed structure.

[0064] To confirm the acyl moiety and determine the stereochemical configuration, chiral GC analysis of the derived methyl 2,3-dihydroxy-3- phenylpropanoate was performed. For this, the isolated compound was saponified using methanol/10% KOH/10% water in a sealed vial at 80⁰C for 1 hour. The acidified solution was extracted with dichloromethane and the dried residue was methylated with diazomethane in diethyl ether at 0⁰C for 5 min. An equivalent volume of water was added and the product was extracted with dichloromethane and concentrated for analysis by chiral GC.

Example Vl.

[0065] Hyoscyamine aldehyde was isolated from the equivalent of 30 yeast expression assays containing (R)-littorine as substrate with no internal standard added (see below). HPLC fractionation of the pooled eluates using an Agilent 100 series HPLC with fraction collector and eluting through a preparative C-18 column (Gemini 5 micron, 250 X 10 mm from Phenomenex ) at 3 ml/min, starting with 50 mM phosphate buffer at pH 9.0 with a linear gradient to 60% acetonitrile in 20 min. This yielded 10 μg of compound judged by HPLC and GC to be greater than 95% pure. GC/MS analysis of the trimethylsilylated compound indicated the presence of primarily one geometric isomer of the enol ether of hyoscyamine aldehyde with molecular ions of m/z 359 and fragment ions of m/z 270 and 124. Reduction of 1 μg of the compound in a 1 mg/ml solution of aqueous NaBH₄ at room temp for 1 min yielded a compound indistinguishable from hyoscyamine by GC/MS (data not shown). The differential decomposition in base suggests that the purified compound is the cis enol (23).

PLANT MATERIALS. Example VII.

[0066] H. niger seeds were provided by Plant Gene Resource of Canada (Saskatoon, Canada). [0067] H. niger and H. muticus plants were grown in soil in a controlled environment chamber with 16 hour /23-24⁰C days and 8 hour /2O⁰C nights under approximately 100 μmol/m²/s light intensity. Cultured roots of H. niger were obtained as reported previously (24) and grown at 25⁰C in the dark on a rotary shaker (100 rpm) in Gamborg B5 medium containing 3% (w/v) sucrose and 1 μM indole-3-butyric acid. Sterile Nicotiana tabacum cv. Xanthi shoots were maintained in vitro (hormone-free MS medium, 3% sucrose, pH 5.8, 0.8% agar).

Example VIII. [0068] H. niger cDNA library construction.

[0069] Total RNA was prepared from leaves or cultured roots of H. niger as described by Carpenter and Simon (25). The polyA⁺ RNA fraction was isolated with the PoIyAT ract^® mRNA Isolation System (Promega, Madison, USA). Double-stranded cDNAs were synthesized and suppression PCR was conducted using the PCR-Select cDNA Subtraction Kit (Clontech) according to the protocol provided by the manufacturer. Double-stranded cDNA from cultured roots and leaves were used as "tester" and "driver," respectively. The resulting PCR products were cloned into the pCRII-TOPO vector (Invitrogen). The resulting plasmids were used to transform ElectroMax™ DH10B™ E. coli cells (Invitrogen) by electroporation. The resulting 1.2 x 10⁵ colonies were pooled and stored as a glycerol stock.

Example IX.

[0070] VIGS in H. muticus.

[0071] For gene silencing studies, the plasmids pTRV1 and pTRV2 (19) were used. pTRV1 encodes tobacco rattle virus RNA1 , and pTRV2 is a VIGS vector based on TRV RNA2. Eight putative cytochrome P450 clones were selected from the H. niger cDNA library and their cDNA inserts were ligated into the vector pTRV2 using appropriate restriction enzymes. pTRV1 and pTRV2 constructs were introduced separately into A. tumefaciens strain C58 by electroporation. The selected transformants were grown overnight at 28° in LB broth supplemented with kanamycin (50 mg/L) and rifampicin (50 mg/L). After centrifugation, bacteria were resuspended in buffer containing 10 mM MES (pH 5.6), 10 mM MgCI₂ and 100 μM acetosyringone to OD₆oo = 1 and allowed to stand at room temperature for 2-4 h.

[0072] Agrobacterium cultures containing pTRV1 and pTRV2 constructs were mixed in 1 :1 ratio and infiltrated into the underside of two or three leaves of eight-week-old H. muticus plants using a 1 ml syringe. Mock infected control plants were infiltrated with Agrobacterium resuspension buffer. Vector control plants were infiltrated with a mixture of Agrobacterium cultures containing pTRV1 and pTRV2 lacking cDNA insert. After 5 weeks, leaf or root material was harvested for alkaloid analysis.

Example X.

[0073] Isolation of full-length HnCYPI cDNA.

[0074] In order to isolate the full-length HnCYPI cDNA, 5' and 3' RACE reactions were performed on polyA⁺ RNA of H. niger cultured roots using the

Marathon™ cDNA Amplification Kit (Clontech). The full-length cDNA was cloned into the pCR2.1-TOPO vector (Invitrogen) and DNA sequences were determined for eight independent clones. One of these clones was designated pRL037.

Example Xl. [0075] RNAi suppression and overexpression of HnCYPI in H. niger and N. tahacum.

[0076] For HnCYPI suppression in H. niger, hairpin RNA expression was used to induce the RNA interference. The cDNA insert of a library clone designated pRLOIO corresponding to the nucleotides 412-923 in the HnCYPI (SEQ ID NO: 1 ) open reading frame was excised with EcoRI and ligated into EcoRI-digested pCR^®8/GW/TOPO^® vector (Invitrogen) to give the Gateway entry clone pRL040. A hairpin RNA expression clone was generated by performing an LR recombination reaction between pRL040 and the Gateway- compatible binary plant RNAi vector pH7GWIWG2(ll) (26) to give pRL041. A. rhizogenes strain ATCC15834 was transformed separately with pRL041 and the empty vector pH7GWIWG2(ll). The bacteria were grown to mid-log phase (OD_6Oo = 0.5) in YMB medium with 100 mg/l spectinomycin at 28⁰C. Cells were centrifuged and resuspended to an OD₆oo of 1.0 in Gamborg B5 medium containing 3% sucrose. Sterile H. niger leaves were cut transversely and incubated with the cells for 5-10 min. The infected leaf pieces were placed on B5 solid medium (0.8% agar) for 48 h and transferred to B5 solid medium supplemented with 500 mg/l carbenicillin. After 7 d, the leaf pieces were transferred to B5 solid medium with 250 mg/l carbenicillin and 10 mg/l hygromycin. Hygromycin-resistant roots emerged within 4 weeks. [0077] The actively growing roots were transferred to 25 ml of B5 liquid medium with 250 mg/l carbenicillin in 125 ml flasks. Individual hairy root lines were derived from separate main roots. The hairy roots were transferred to B5 medium with decreasing concentrations of carbenicillin (100, 50 and 0 mg/l) each week. For alkaloid production, transformed hairy roots were maintained in 250 ml flasks with 50 ml B5 medium and shaken at 100 rpm on a rotary shaker at 25⁰C in the dark. Each line was cultured in triplicate for alkaloid analysis. [0078] For HnCYPI overexpression in H. niger, a Gateway entry clone (pRL038) was prepared by PCR amplification of the HnCYPI ORF (Open Reading Frame) from pRL037 (using Pfu DNA polymerase (Stratagene) and the oligonucleotides 5^I-ACCATGTATATTGAAG-ATACAAGTGAAATC-3^• (SEQ ID NO: 3) and δ'-TTATGAGTTCCT-AATTTTTGGTATG-S) (SEQ ID NO: 4) and cloning of the product into the pCR^®8/GW/TOPO^® vector (Invitrogen). An expression clone was generated by performing an LR recombination reaction between the entry done pRL038 and a Gateway-compatible binary vector pMDC32 (27) to give pRL042 as shown in FIG. 10. A. rhizogenes strain ATCC15834 was transformed separately with pRL042 and pMDC32 and grown in YMB medium with 50 mg/l kanamycin at 28⁰C. Transformed H. niger hairy roots were produced as described above.

[0079] A. rhizogenes carrying empty vector pMDC32 or pRL042 were also used to infect tobacco leaf disks and hairy roots were produced as described for H. niger, except that the transformed roots were cultured in the presence of 20 mg/l hygromycin.

[0080] The metabolism of littorine enantiomers in transgenic hairy roots was studied as follows. About 0.4 g of tobacco hairy roots were subcultured in 10 ml of B5 medium containing 3% sucrose in 50 ml flasks for 3 d. The cultures were supplied separately with solutions (R)- and (S)-littorine in ethanol to a final concentration of 0.1 mM. After 3 d, the roots were harvested for alkaloid analysis.

Example XII. [0081] Analysis of HnCYPI gene expression by RT-PCR.

[0082] For gene expression analysis, total RNA was isolated from different plant tissues using RNeasy Plant Mini Kit (Qiagen), and on-column DNase digestion with RNase-free DNase Set (Qiagen) was performed for each sample during RNA isolation according to the protocol provided by the manufacturer. ThermoScript™ reverse transcriptase and random hexamer primers (Invitrogen) were used to synthesize cDNA. cDNA was amplified by PCR using an Advantage 2 Polymerase Mix (Clontech) and the following gene- specific primers: roots of VIGS H. muticus plants, 5'- CTCATAAAGCTGTTGAATCACAAGTG-3' (forward) (SEQ ID NO: 5) and 5'- CATACCTTCACCTATACCTTTGCCTTCA-3' (reverse) (SEQ ID NO: 6); all other plant tissues, δ'-CACAGTTGAATGGACATTGGTGGAGC-S' (forward) (SEQ ID NO: 7) and δ'-GAACAGTAATGGCGCCGGAGGATGC-S' (reverse) (SEQ ID NO: 8). The 18S rRNA primer-competimer mix (universal 18S internal standards kit; Ambion) was used as an internal standard in multiplex PCR experiments. The PCR products were visualized on 2% agarose gels containing ethidium bromide.

Example XIII.

[0083] Characterization of HnCYPI expressed in yeast. [0084] A yeast expression clone was generated by performing an LR recombination reaction between the entry clone pRL038 and a Gateway destination vector pYES-DEST52 (Invitrogen) to give pRL039. Yeast transformations were performed on the WAT11 strain (28), using the S. c. EasyComp™ Transformation Kit (Invitrogen). A transformation with the empty vector pYES-DEST52 was performed as a control. Transformants were selected by growth on synthetic complete medium lacking uracil (SC-ura) and containing 2% glucose. The colonies were transferred into 10 ml of SC-ura liquid medium containing 2% glucose and grown at 28⁰C for 24 h. The overnight cultures were used to inoculate 200 ml of YPGE medium (10 g/l yeast extract (Difco), 10 g/l bactopeptone (Difco), 5 g/l glucose, 3% (v/v) ethanol) and cells were grown at 28⁰C until cell density reached 8 x 10⁷ cells per ml. Induction was started by the addition of galactose to 2% (w/v). The induction was continued for about 12-16 h until the cell density reaches 2-5 x 10⁸ cells per ml. Cells were harvested and washed with 25 ml of washing buffer having 100 mM potassium phosphate buffer, pH 7.5, 1 mM EDTA₁ 0.1 M KCI. Cells were resuspended in 3 ml of isolation buffer containing 100 mM potassium phosphate buffer, pH 7.5, 5 mM EDTA, 2 mM dithiothreitol, 250 mM sucrose, 1 mM phenylmethylsulphonyl fluoride. Yeast microsomes were prepared according to Katavic et al. (29) and the microsomal membrane pellet was resuspended in storage buffer containing 100 mM potassium phosphate buffer, pH 7.5, 1 mM EDTA, 20% (v/v) glycerol. Protein concentration was determined by Bradford assay (Bio-Rad).

[0085] For enzyme assays, the complete reaction mixture (200 μl) contained 200-400 μg microsomal protein, 100 mM potassium phosphate buffer, pH 7.4, 3 mM NADPH, an NADPH regenerating system (6.7 mM glucose 6- phosphate and 0.4 IU of glucose-6-phosphate dehydrogenase), and alkaloid substrate (865 μM unless otherwise stated). Reactions were initiated by the addition of NADPH and carried out at 3O⁰C for 4-180 min with gentle shaking. After stopping the reactions by adding 100 μl of 30% NH₄OH, 3.5 μg of scopolamine (Sigma) was added as internal standard. The reaction mixtures were applied to Extrelut QE columns (EM Science Gibbstown, NJ). After 15 min, the alkaloids were eluted with 10 ml of dichloromethane and the solvent was removed under a nitrogen stream. For GC or GC/MS analysis, the residue was dissolved in 20 ul of BSA/pyridine (1 :1 ). For kinetic analysis, HnCYPI was assayed using (R)-littorine at concentrations ranging from 17 to 277 μM. Reactions were initiated by the addition of NADPH and carried out at 30⁰C for 8 min. The K_m was calculated from a double reciprocal plot of the initial velocity versus substrate concentration from triplicate data.

RESULTS Example XIV.

[0086] Expressed sequence tags from H. niger.

[0087] Given the relatively limited knowledge of the genes involved in the biosynthesis, transport and regulation of tropane alkaloids in the Solanaceae, a collection of expressed sequence tags from H. niger was assembled as an aid to address this issue. Since alkaloid biosynthesis in H. niger occurs in the roots (1), a cDNA library was constructed to enhance the frequency of cDNAs corresponding to genes expressed root-specifically. For this subtracted library, cDNA from H. niger root cultures was used as the tester and leaf cDNA was used as the driver in a suppression PCR procedure. To generate ESTs from the library, bacterial colonies representing cDNA clones were picked randomly and their plasmid inserts were sequenced. After sequence analysis, the 2,286 EST sequences representing 214 clusters and 1 ,299 singletons were subjected to similarity searches using BLAST.

[0088] To determine whether a cytochrome P450 was involved in the conversion of littorine to hyoscyamine, the H. niger ESTs showing similarity to cytochrome P450s were studies. Twenty five ESTs (4 clusters and 11 singletons) showed similarity to cytochrome P450 sequences in the CYP families 71 , 73, 74, 76, 80, 82 and 86. Eight cDNA clones representing these families were chosen as candidates for functional identification using VIGS.

Example XV.

[0089] Suppression of Hyoscyamus cytochrome P450s by virus- induced gene silencing.

[0090] A TRV-based VIGS system was found to be an effective tool for transient loss-of function experiments in Hyoscyamus. Proof-of-concept studies showed that TRV-mediated silencing of phytoene desaturase was more effective in H. muticus (Egyptian henbane) than in H. niger, and consequently the former species was used as the VIGS host plant. The two Hyoscyamus species have similar leaf alkaloid levels and profiles (data not shown). The silencing of several known genes in tropane alkaloid biosynthesis in H. muticus, as evidenced by reduced alkaloid levels in infected plants (unpublished), demonstrated that recombinant TRV could successfully silence the tropane alkaloid pathway in roots of this species.

[0091] For functional identification, the DNA inserts of eight selected cDNA clones showing similarity to cytochrome P450s were subcloned into the VIGS binary vector pTRV2 (19). The eight VIGS plasmids containing CYP inserts were used separately to transform A. tumefaciens, then mixed with A. tumefaciens containing pTRVL Gene silencing was effected by infiltration of the resulting mixtures separately into the leaves of H. muticus plants. Negative (mock infection and empty vector) and positive (phytoene desaturase) controls were also performed. After five weeks, alkaloids were extracted from leaf material and analyzed by GC. [0092] For seven of the eight cytochrome P450 VIGS constructs, no significant difference in the leaf alkaloid profiles was detectable (data not shown). Negative and positive control experiments showed no change in alkaloid profiles in plants subjected to VIGS (see FIG. 3). Vector control plants appeared healthy and plants undergoing silencing of the phytoene desaturase gene showed the expected bleached phenotype (not shown). Significantly, silencing of the gene corresponding to a cDNA library clone called pRL011 using the VIGS construct pRL033 had a profound and reproducible effect on alkaloid quality and quantity as shown in FIG. 3A. In this case, total tropane alkaloid content, and notably that of hyoscyamine and scopolamine was decreased, yet littorine levels increased in the same plants. Since littorine is thought to be a precursor of hyoscyamine, this suggested a role for the cytochrome P450 gene corresponding to pRL011 in the conversion of littorine to hyoscyamine.

Example XVI.

[0093] Cloning and sequence analysis of full length H. niger HnCYPI cDNA.

[0094] The cDNA clone pRL011 is one of five ESTs which were found to be similar to the plant CYP80B subfamily members. Based on sequence data in comparison with a full length cDNA (see below), the five clones appeared to represent Rsal restriction fragments of cDNAs of the same gene.

[0095] A full length clone, pRL037, corresponding to the partial ORF of pRL011 was obtained by 5'and 3' RACE and PCR amplification. The cDNA insert of 1745 nt (SEQ ID NO: 1 ) has an open reading frame of 510 amino acids (SEQ ID NO: 2) predicting a polypeptide of molecular mass 58.6 kDa. The predicted amino acid sequence shares 41 , 39 and 38% sequence identity with the Coptis japonica, Papaver somniferum and Eschscholzia californica (S)-N- methylcoclaurine-3'-hydroxylases, respectively (30, 31). These enzymes are in the CYP80B subfamily and hydroxylate an aromatic position of an Λ/-methylated intermediate in the benzylisoquinoline alkaloid pathway. The H. niger enzyme was named HnCYPI . Based on enzyme activity (see below), the name littorine mutase/monooxygenase is proposed. HnCYPI also shares 30-35% identity with various members of the CYP75, CYP76 and CYP80 families in the CYP71 clan of cytochrome P450's. The relationship among sequences related to HnCYPI is illustrated in the phylogenetic tree shown in FIG. 3. In terms of known enzymes, these include flavonoid hydroxylases, geraniol hydroxylase, ethoxycoumarin deethylase and berbamunine synthase. The latter enzyme couples (S)-/V-methylcoclaurine with a second Λ/-methylcoclaurine enantiomer and thus also utilizes /V-methylated alkaloid substrates (32).

Example XVII.

[0096] Suppression by RNA interference and overexpression of HnCYPI in H. niger.

[0097] To confirm the VIGS results and obtain propagable plant tissue in which HnCYPI is suppressed, RNA interference experiments in hairy roots of H. niger were carried out. As shown in FIG. 3B, suppression of HnCYPI by RNAi gives a phenotype similar to that observed for VIGS experiments with HnCYPI . This is similar to the observed effects of cytochrome P450 inhibitors on alkaloid accumulation in Datura stramonium hairy roots (35) except that littorine levels are not significantly reduced in H. niger hairy roots (FIG. 3B). In addition, a compound identified as 3'-hydroxylittorine (see below), which was found to be present in control roots, showed reduced levels in roots undergoing HnCYPI gene silencing.

[0098] In similar studies, H. niger hairy root cultures were generated in which HnCYPI was overexpressed. While competitive RT-PCR indicated modest overexpression of HnCYPI of up to approximately 3-fold relative to wild type (data not shown), no significant change in alkaloid content or profile was observed. Consequently, it appears that HnCYPI does not limit the accumulation of alkaloid under the conditions tested.

Example XVIII.

[0099] Tissue specific expression of HnCYPI . [00100] HnCYPI showed a pattern of gene expression consistent with its involvement in tropane alkaloid biosynthesis. FIG. 5A shows the results of competitive RT-PCR indicating the root-specific expression of HnCYPI . Similar RT-PCR experiments demonstrate the resulting reduction in root expression of HnCYPI upon VIGS using pRL033 in four different plants confirmed the desired gene silencing effect (FIG. 5B).

Example XIX.

[00101] Expression of HnCYPI in tobacco hairy roots. [00102] The function of HnCYPI was tested by expression in N. tabacum hairy roots which are normally devoid of tropane alkaloids. The HnCYPI open reading frame was cloned into the vector pMDC32 and used to generate N. tahacum hairy root cultures. FIG. 6 shows the results of alkaloid analysis for vector control cultures and hairy roots expressing HnCYPI when the culture medium was supplemented with (R)-littorine. A compound which eluted slightly earlier than littorine was evident in littorine-supplemented cultures expressing HnCYPI . This was identified by GC/MS as hyoscyamine based on comparison with a standard (data not shown). This conversion was stereospecific in the sense that similar experiments with (S)-littorine did not yield detectable product (data not shown). This confirms the VIGS and RNAi results indicating that HnCYPI is involved in littorine-to-hyoscyamine conversion. Indeed, it suggests the possibility that HnCYPI may convert littorine directly to hyoscyamine. Other possibilities include catalysis of more than one step by HnCYPI or the possible involvement of one or more N. tabacum enzymes in a multiple step conversion.

Example XX.

[00103] Characterization of HnCYPI in yeast.

[00104] The activity of HnCYPI was characterized in detail by expression in S. cerevisiae strain WAT11 , which has been engineered to express a plant cytochrome P450 reductase (28). This revealed a fairly complex pattern of products from different alkaloid substrates. Some of this complexity results from enzyme activities found in wild-type yeast microsomes. [00105] The yeast strain WAT11/pRL039 yielded microsomes which had readily measurable enzyme activity on certain tropane alkaloids. FIG. 7B shows a gas chromatogram of the trimethylsilylated alkaloid products of HnCYPI- containing yeast microsomes incubated with (R)-littorine and NADPH. [00106] When compared to products of control microsomes (FIG. 7A), a number of novel peaks are evident. Production of the corresponding compounds was found to be dependent on NADPH, but not NADH (data not shown). Clotrimazole, a CYP inhibitor which has been found to block littorine- to-hyoscyamine conversion in Datura stramonium roots (35), inhibited the reaction in yeast microsomes by 50% at a concentration of 0.1 mM (data not shown). The reaction was not found to be stimulated by S-adenosylmethionine (data not shown).

[00107] As numbered in FIG. 7B, Peak 1 was identified as phenylacetoxytropane by GC/MS (m/z(%):259 (M+, 8), 140 (8), 124 (100), 91 (21 ), 82 (27), 42 (12)) in comparison to published MS data (36). Peaks 2 and 3 were identified as trimethylsilylhomatropine and trimethylsilylhyoscyamine, respectively, by GC/MS comparison with trimethylsilylated commercial standards (data not shown). Peaks 5 and 6 were assigned to the geometric isomers of the trimethylsilyl derivatives of the enol form of hyoscyamine aldehyde. GC/MS analysis indicated a molecular ion of 359 m/z and fragment ions assignable to an unmodified tropine moiety. NaBH₄ reduction yielded a compound with the GC/MS properties of hyoscyamine .

[00108] Peak 7 was identified as the trimethylsilyl derivative of (2'R,3'R)- 3'-hydroxylittorine. The GC retention time and mass spectrum of peak 7 was identical to derivatized 3'-hydroxylittorine isolated and characterized from H. niger roots (see below). Also, methyl (27?,3'R)-dihydroxy-3-phenylpropanoate was detected by chiral GC/MS after hydrolysis and methylation of extracts of the microsomal assay.

[00109] Peaks 9 and 10 were not assigned, but based on GC/MS appear to be tropanol derivatives.

[00110] When (S)-littorine was a substrate, the only product detected was 3'-hydroxylittorine that was identified as the 2'S,3'S isomer when the derived methyl ester was compared to standards using chiral GC (data not shown).

[00111] Thus, the major products of HnCYPI from (R)-littorine are (2'f?,3'/^:?)-3'-hydroxylittorine and hyoscyamine aldehyde. The latter compound has not been previously reported as a natural product. However, the para hydroxy derivative of littorine (4'-hydroxylittorine, using an alternative carbon numbering, or 3α-(p-hydroxphenyl)lactoyloxytropane) has been reported to occur in a number of Hyoscyamus species (37, 38). Based on the reported mass spectrum, it is possible that this compound is actually 3'-hydroxylittorine. [00112] Other products of the HnCYPI enzyme assay included the naturally occurring alkaloids hyoscyamine, homatropine and phenylacetoxytropane (39). Given that hyoscyamine was the major product of (R)-littorine from tobacco expression of HnCYPI , the yeast expression results showing multiple products required additional investigation. [00113] To determine if some of the HnCYPI products might be derived from others, and additionally to test the possible involvement of yeast enzymes and non-enzymatic reactions, hyoscyamine aldehyde, 3'-hydroxylittorine and hyoscyamine were tested as substrates in the microsomal system. When hyoscyamine aldehyde was mixed with control yeast microsomes after the addition of NH₄OH, homatropine and 3-phenylacetoxytropane were detected. Thus, it appears that under the conditions of the assay, these compounds are produced non-enzymatically (23). When hyoscyamine aldehyde was incubated with control yeast microsomes, hyoscyamine, as well as homatropine and 3- phenylacetoxytropane, was detected. HnCYPI -containing microsomes also yielded these three products, as well as a novel compound (peak 8 in FIG. 7D). This was tentatively identified by GC/MS as 2'-hydroxyhyoscya^'mine, a tropane alkaloid recently reported to occur in Datura stramonium (40).

[00114] Hyoscyamine itself was also tested as a substrate in yeast microsomes (data not shown). Interestingly, most of the (R)-littorine products also appear to be produced from hyoscyamine. Indeed, all of the products of (R)-littorine listed above, with the exception of 3'-hydroxylittorine (i.e., hyoscyamine aldehyde, 2'-hydroxyhyoscyamine, homatropine, 3- phenylacetoxytropane), are produced from hyoscyamine in a HnCYPI- dependent fashion.

[00115] Unlike hyoscyamine, no products of HnCYPI were detected from 3'-hydroxylittorine (data not shown). In order to test this, a source of 3'- hydroxylittorine was required. Analysis of H. niger roots indicated that a compound with the expected properties of 3'-hydroxylittorine was present. One hundred and twenty five μg were isolated and characterized (see Methods).

This compound represents a novel plant natural product whose occurrence tends to confirm the validity of the yeast microsome results implicating 3'- hydroxylittorine as a product of HnCYPI .

[00116] Based on measurements with (R)-littorine conversion to 3'- hydroxylittorine, the pH optimum and (R)-littorine K_m for HnCYPI were determined to be 7.2 and 34 μM, respectively.

[00117] Although the present invention has been shown and described with respect to various exemplary embodiments, various additions, deletions, and modifications that are obvious to a person of ordinary skill in the art to which the invention pertains, even if not shown or specifically described herein, are deemed to lie within the scope of the invention as encompassed by the following claims. Further, features or elements of different embodiments may be employed in combination.

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Dinesh-Kumar, S. P., Liu, Y. and Schiff, M., international patent application PCT/US03/08410.

Claims

CLAIMS What is claimed is:

1. An isolated or recombinant nucleic acid encoding a littorine mutase/hydroxylase.

2. The isolated or recombinant nucleic acid of claim 1 , wherein the nucleic acid is of a Hyoscyamus niger origin.

3. The isolated or recombinant nucleic acid of claim 1 or claim 2, wherein the littorine mutase hydroxyase comprises SEQ ID NO: 2.

4. The isolated or recombinant nucleic acid of any one of claims 1-3, wherein a nucleotide sequence of the isolated or recombinant nucleic acid is selected from the group consisting of SEQ ID NO: 1 ; a nucleotide sequence that hybridizes to SEQ ID NO: 1 , and fragments of either thereof.

5. A vector comprising the isolated or recombinant nucleic acid of any one of claims 1-4.

6. The vector of claim 5, wherein the nucleic acid encoding the littorine mutase/hydroxylase is operably coupled to a promoter.

7. The vector of claim 6, wherein the promoter is temporal, tissue specific or constitutive.

8. The vector of any one of claims 5-7, wherein the vector is an expression vector or a cloning vector.

9. The vector of any one of claims 5-8, wherein the nucleic acid encoding the littorine mutase/hydroxylase is associated with a marker gene.

10. A cell transformed with the vector of any one of claims 5-9.

11. Use of the cell of claim 10 for the production of a tropane alkaloid.

12. Use of the cell of claim 11 , wherein the tropane alkaloid is selected from the group consisting of phenylacetoxytropane, trimethylsilylhomatropine, trimethylsilylhyoscyamine, a trimethylsilyl derivative of (2'R,3'R)-3^r- hydroxylittorine, methyl (2'R,3'R)-dihydroxy-3-phenylpropanoate, 3'- hydroxylittorine, (2'R,3'R)-3'-hydroxylittorine, hyoscyamine aldehyde, hyoscyamine, homatropine, phenylacetoxytropane, 3-phenylacetoxytropane, and 2'-hydroxyhyoscyamine.

13. The cell of claim 10, wherein the cell comprises a microbial host cell.

14. The cell of claim 13, wherein the microbial host cell is a bacterial cell or a yeast cell.

15. The cell of claim 13, wherein the microbial host cell is selected from the group consisting of Agrobacterium tumafaciens, Saccharomyces cerevisiae, Escherichia coli, and Agrobacterium rhizogenes.

16. The cell of claim 10 wherein the cell is a plant cell.

17. A transgenic plant, plant seed or progeny thereof transformed with the isolated or recombinant nucleic acid of any one of claims 1-4 or the vector of any one of claims 5-9.

18. The transgenic plant, plant seed or progeny thereof of claim 17, wherein the transgenic plant, plant seed or progeny thereof is of a Solanaceae origin.

19. The transgenic plant, plant seed or progeny thereof of claim 17, wherein the transgenic plant, plant seed or progeny thereof is selected from the group consisting of Hyoscymus muticus, Hyoscymυs niger, and Nicotinia tabacum.

20. A process for cultivating a transgenic plant, plant seed or progeny thereof, comprising: planting the transgenic plant, plant seed or progeny thereof of any one of claims

17-19; and growing the transgenic plant, plant seed or progeny thereof to maturity.

21. The process according to claim 20, further comprising harvesting tissue from the mature transgenic plant, plant seed or progeny thereof.

22. The process according to claim 20 or claim 21 , further comprising isolating the littorine mutase/hydroxylase from the transgenic plant, plant seed, progeny thereof, or harvested tissue.

23. An isolated littorine mutase/hydroxylase produced by the process according to claim 22.

24. An isolated antibody or fragment thereof capable of binding to the isolated littorine mutase/hydroxylase of claim 23.

25. The process according to any one of claims 20-22, further comprising producing an intermediate product including the tropane alkaloid from the transgenic plant, plant seed, progeny thereof, or harvested tissue.

26. An intermediate product including the tropane alkaloid produced by the process according to claim 25.

27. Use of the isolated tropane alkaloid ot claim 26 tor the manufacture of a medicament for the treatment or administration of the tropane alkaloid to a subject in need thereof.

28. The isolated tropane alkaloid of claim 26 or claim 27, wherein the tropane alkaloid is selected from the group consisting of hyoscyamine, scopolamine and 3-(3-phenylglyceroloxy)tropane.

29. An RNAi agent specific for the isolated or recombinant nucleic acid of any one of claims 1-4.

30. Use of the RNAi agent of claim 29 to block one or more steps of a tropane alkaloid biosynthesis pathway in a plant, plant cell or progeny thereof.

31. Use of the RNAi agent of claim 30, wherein the plant, plant cell or progeny thereof is selected from the group consisting of Hyoscymus muticus, Hyoscymus niger, and Nicotinia tabacυm.

32. A process for producing a precursor or intermediate compound of a tropane alkaloid biosynthesis pathway, comprising: planting the transgenic plant, plant seed or progeny thereof of any one of claims

17-19; blocking one or more steps of a tropane alkaloid biosynthesis pathway with an RNAi agent specific for a nucleic acid encoding a littorine mutase/hydroxylase; growing the transgenic plant, plant seed or progeny thereof to maturity; and isolating the precursor or intermediate compound of the tropane alkaloid biosynthesis pathway from a tissue of the transgenic plant, plant seed or progeny thereof.

33. An isolated precursor or intermediate compound of the tropane alkaloid biosynthesis pathway produced by the process of claim 32.

34. Use of the isolated precursor or intermediate compound of claim 33 for the manufacture of a medicament for the treatment or administration of the tropane alkaloid to a subject in need thereof.