WO2007112596A1 - Nucleotide sequences encoding enzymes in biosynthesis of dihydroartemisinic acid - Google Patents

Abstract

Isolated nucleic acid molecules cloned from Artemisia annua encode artemisinic aldehyde double bond reductase and artemisinic/dihydroartemisinic aldehyde dehydrogenase. Artemisinic aldehyde double bond reductase enzymatically reduces artemisinic aldehyde to dihydroartemisinic aldehyde. Artemisinic/dihydroartemisinic aldehyde dehydrogenase enzymatically oxidizes dihydroartemisinic aldehyde to dihydroartemisinic acid and artemisinic aldehyde to artemisinic acid. The nucleic acid molecules, and the enzymes encoded thereby, may be used in processes to produce dihydroartemsinic aldehyde, dihydroartemisinic acid or artemisinic acid in a host cell. Dihydroartemisinic acid is a late precursor to the antimalarial compound artemisinin.

Description

NUCLEOTIDE SEQUENCES ENCODING ENZYMES IN BIOSYNTHESIS OF

DIHYDROARTEMISINIC ACID

Cross -reference to Related Applications

[0001] This application claims the benefit of United States Provisional Patent

Applications USSN 60/789,138 filed April 5, 2006 and USSN 60/857,503 filed November 8, 2006, both of which are herein incorporated by reference in their entirety.

Field of the Invention

[0002] The present invention relates to production of plant-derived compounds of health and commercial interest. More particularly, the present invention relates to nucleotide sequences encoding enzymes, to enzymes encoded by the nucleotide sequences and to processes for producing (:/7S)-dihydroartemisinic aldehyde, ( 11 R)- dihydroartemisinic acid and/or artemisinic acid therewith.

Background of the Invention

[0003] Plants, in general, contain a myriad of secondary metabolites often synthesized by unique biochemical processes operating only in exotic species. For plant- derived products such as drugs, the 1997 worldwide sales were US$ 10 billion (Rotheim 2002). In many cases the supply of the relevant plant material for these drugs is limited or variable. One approach to developing methods for producing these drugs is to apply the methods of biochemistry, molecular biology and genomics to elucidate the biosynthesis and relevant biosynthetic genes for compounds of value for human health.

[0004] With the realization that many of the enzymes involved in natural product biosynthesis represent variations within known classes of enzymes, expressed sequence tag (EST) analysis (combined with heterologous expression) provides a powerful means of identifying their corresponding genes (Cahoon et al. 1999, Gang et al. 2001 , Lange et al. 2000 and van de Loo et al. 1995)

[0005] One area of interest is bioactive compounds of the tribe Anthemideae in the family Asteraceae (Compositae) (Torrell et al. 1999 and Watson et al. 2000). Anthemideae (Asteraceae, subfamily Asteroideae) is a tribe of 109 genera which includes daisies, chrysanthemums, tarragon, chamomile, yarrow and sagebrushes (Watson et al. 2000). These plants are aromatic in nature resulting from high concentrations of mono- and sesqui- terpenes. Many of the species in this tribe are valued for the health benefits or insecticidal properties. [0006] Of particular interest is artemisinin from Artemisia annua or sweet wormwood. In 1972, Chinese scientists isolated the sesquiterpene lactone containing an endoperoxide group (see Fig. 1 ) from Artemisia and called it qinghaosu (van Agtmael et al. 1999b). Prior to this sweet wormwood or qinghao had been used in traditional Chinese medicine for centuries. Artemisinin has become very important for the treatment of malaria in Southeast Asia and elsewhere, particularly for multi-drug-resistant falciparum forms of the disease (O'Neill 2005, Rathore et al. 2005, Robert et al. 2002, Wilairatana et al. 2002 and Wu 2002). Since the discovery of artemisinin, a number of semi-synthetic derivatives have been developed for specific applications in malaria treatment.

[0007] Malaria remains a serious health problem which affects over 400 million people, especially in Africa and Southeast Asia, causing the deaths in excess of 2 million each year. Increasing resistance of the malaria parasite, Plasmodium falciparum, towards current antimalarial drugs is a cause for concern. The future value of antimalarial drugs based on the artemisinin structure is illustrated by the development by Bayer AG of Artemisone, an artemisinin derivative reported to be 10-30 fold more active than artesunate, for which clinical trials are currently under way. Also, researchers at the Walter Reed Army Institute of Research (USA) are currently developing artelinic acid for intravenous treatment of severe malaria.

[0008] Artemisinin is produced in relatively small amounts of 0.01 to 1.0% dry weight, making it and its derivatives relatively expensive (Gupta et al. 2002). Several studies describe the chemical synthesis of the sesquiterpene, but none are an economical alternative for isolation of artemisinin from the plant (Yadav et al. 2003). Therefore a higher concentration in the plant or production in an alternative host is desirable to make artemisinin available as economically as possible, especially for use in the Third World. Knowledge of the biosynthetic pathway and the genes involved should enable engineering of improved production of artemisinin. Alternatively, there is also the possibility of producing intermediates in the pathway to artemisinin which are of commercial value. For example, a compound 15 times more potent in vitro than artemisinin against Plasmodium falciparum has been synthesized from artemisinic alcohol (Jung et al. 2001 ).

[0009] There is evidence that artemisinin is localized to glandular trichomes on the surfaces of certain tissues of the plant (Duke et al. 1994 and Duke et al. 1993). The number and even existence of these trichomes and the amount of artemisinin varies widely among biotypes. [0010] Typically, compounds discovered in plants and found to be useful are produced commercially by i) chemical synthesis, where possible and economical, ii) extraction of cultivated or wild plants, or iii) cell or tissue culture (this is rarely economical). In those cases in which chemical synthesis is not economical, it makes sense to learn as much as possible about the biosynthesis of a natural product, such that it can be produced most efficiently in plants or cell/tissue culture. In the case of artemisinin, chemical synthesis is not commercially feasible. Since the compound is produced in small quantities in Artemisia, the drugs derived from artemisinin are relatively expensive, particularly for the Third World countries in which they are used. While the antimalarial drugs, chloroquine and sulfadoxine-pyrimethamine, cost as little as 20 cents for an adult treatment, artemisinin-derived compounds, by contrast, can be 100 times as expensive. Chloroquine resistance is prevalent and sulfadoxine-pyrimethamine resistance is increasing. The World Health Organization recently added the artemisinin-derived drug, artemether to their Model List of Essential Medicines, which are recommended to be available at all times in adequate amounts and in the appropriate dosage forms, and at a price that individuals and the community can afford. Consequently, it would be useful to be able to supply artemisinin-derived drugs more economically.

[0011] There are numerous patents relating to artemisinin and artemisinin derived drugs. These cover drug synthesis and formulation, Artemisia cultivation (Kumar 2002) and tissue culture and artemisinin extraction (Elferaly 1990). Commonly owned United States patent application 60/729,210 filed October 24, 2005, the disclosure of which is herein incorporated by reference, and now filed as a PCT patent application, discloses a gene encoding amorpha-4,11-diene hydroxylase, which catalyzes the first committed steps in artemisinin biosynthesis (Fig. 1 ).

[0012] In the past five years a reasonably clear picture of artemisinin biosynthesis has emerged as illustrated in Fig. 1 (Bertea et al. 2005). The identity of amorpha-4,11- diene as a biosynthetic intermediate was established, based on the presence of trace of amorpha-4,11-diene in Artemisia extracts and the cloning and expression of cDNAs representing amorpha-4,1 1-diene synthase, a terpene cyclase (Bouwmeester et al. 1999 and Wallaart et al. 2001 ). A cytochrome P450 gene designated cyp71av1 was recently cloned and characterized (Teoh et al. 2006). The cyp71av1 gene encodes a hydroxylase that catalyzes the conversion of amorpha-4,11-diene to artemisinic alcohol. CYP71AV1 expressed in yeast is also capable of oxidizing artemisinic alcohol to artemisinic aldehyde and artemisinic aldehyde to artemisinic acid. Summary of the Invention

[0013] The invention described herein addresses the production of artemisinin and artemisinin-related compounds, including precursors, of pharmaceutical and commercial interest.

[0014] There is provided an isolated nucleic acid molecule comprising a nucleotide sequence having at least 70% nucleotide sequence identity to SEQ ID No.: 3 and encoding an artemisinic aldehyde double bond reductase.

[0015] There is provided an isolated nucleic acid molecule comprising a nucleotide sequence having at least 70% nucleotide sequence identity to SEQ ID No.: 7 and encoding an artemisinic/dihydroartemisinic aldehyde dehydrogenase.

[0016] There is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding an artemisinic aldehyde double bond reductase having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 2.

[0017] There is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding an artemisinic/dihydroartemisinic aldehyde dehydrogenase having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 6.

[0018] There is provided a purified artemisinic aldehyde double bond reductase having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 2.

[0019] There is provided a purified artemisinic aldehyde double bond reductase having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 6.

[0020] There is provided a use of one or more isolated nucleic acid molecules of the present invention in the production of (^S)-dihydroartemisinic aldehyde, {11 R)- dihydroartemisinic acid and/or artemisinic acid.

[0021] There is provided a use of one or more of a purified artemisinic aldehyde double bond reductase or artemisinic/dihydroartemisinic aldehyde dehydrogenase encoded by one or more isolated nucleic acid molecules of the present invention in the production of (Y tS)-dihydroartemisinic aldehyde, (^■/^■/R)-dihydroartemisinic acid and/or artemisinic acid. [0022] There is provided a process for producing (77S)-dihydroartemisinic aldehyde,

( 77R)-dihydroartemisinic acid and/or artemisinic acid comprising expressing or overexpressing one or more isolated nucleic acid molecules of the present invention in a host cell.

[0023] There is provided a process for producing ( 77S)-dihydroartemisinic aldehyde,

(77R)-dihydroartemisinic acid and/or artemisinic acid comprising producing or overproducing an artemisinic aldehyde double bond reductase and/or artemisinic/dihydroartemisinic aldehyde dehydrogenase of the present invention in a host cell.

[0024] The isolated nucleic acid molecules are preferably derived from A. annua.

[0025] Overexpression of one or more of the nucleic acid molecules or overproduction of the artemisinic aldehyde double bond reductase and/or artemisinic/dihydroartemisinic aldehyde dehydrogenase may be done in A. annua. Expression of one or more of the nucleic acid molecules or expression of the artemisinic aldehyde double bond reductase and/or artemisinic/dihydroartemisinic aldehyde dehydrogenase may be done in other hosts, for example plants, yeasts or bacteria. Overexpression or expression of one or more of the isolated nucleic acid molecules of the present invention may be done in combination with overexpression or expression of one or more other nucleic acid molecules involved in the biosynthesis of artemisinin, for example amorpha-4,11-diene synthase and/or amorpha-4,11-diene hydroxylase.

[0026] Part of the solution to the problem of producing artemisinin in an economical and timely fashion is the isolation and exploitation of genes involved in artemisinin biosynthesis. As in other examples of metabolic engineering, such genes can be used to enhance production by overexpression in the native plant (A. annua), a different plant, or in micro-organisms such as bacteria or yeast. An example of this is the expression of the amorphadiene synthase gene in E. coli to produce the artemisinin precursor amorphadiene (Martin et al. 2003) and the production of artemisinic acid in yeast (Ro et al. 2006). Two important steps in the pathway to artemsinin per se, are the reduction of artemisinic aldehyde to (77R)-dihydroartemisinic aldehyde and the oxidation of (77R-dihydroartemisinic aldehyde to (77R)-dihydroartemisinic acid. Consequently, the genes involved in these steps may be used to produce (77R)-dihydroartemisinic acid in a host, alone or in combination with each other and/or with one or more of amorphadiene synthase and amorphadiene hydroxylase. [0027] The resulting (77ft)-dihydroartemisinic acid could then be chemically converted to artemisinin or related compounds of commercial value. Dihydroartemisinic acid is the presumed immediate precursor of artemisinin, and its transformation to artemisinin has been shown to occur spontaneously through photo-oxidation, requiring no enzyme intervention (Sy et al. 2002 and Wallaart et al. 1999). Consequently, using (11 R)- dihydroartemisinic acid instead of artemisinic acid as the starting material for semi-synthesis of artemisinin reduces the number of steps required for artemisinin production thus, simplifying the production process. This may lead to shorter artemisinin production time and lower production cost. The eventual outcome will be cheaper artemisinin and artemisinin- related drugs. Alternatively, (77S)-dihydroartemsinic acid could be chemically converted to (77S)-artemisinin which is expected to have antimalarial activity.

[0028] The genes (nucleic acid molecules) of the present invention may be derived, for example cloned, from Artemisia annua. Cloned nucleic acid molecules were sequenced and characterized by expression in E. coli. One of the cloned nucleic acid molecules encodes a double-bond reductase which reduces the C11-C13 double bond of artemisinic aldehyde to form (77S)-dihydroartemisinic aldehyde as the major product. Another of the cloned nucleic acid molecules encodes an aldehyde dehydrogenase for the conversion of dihydroartemisinic aldehyde to dihydroartemisinic acid. The aldehyde dehydrogenase is further capable of dehydrogenating artemisinic aldehyde to artemisinic acid.

[0029] Nucleic acid molecules of the present invention may also be used in the development of DNA markers and in targeted mutagenesis techniques (e.g. TILLING (Targeting Induced Local Lesions IN Genomes)).

[0030] A genetic marker (DNA marker) is a segment of DNA with an identifiable physical location on a chromosome and associated with a particular gene or trait and whose inheritance can be followed. A marker can be a gene, or it can be some section of DNA with no known function. Because DNA segments that lie near each other on a chromosome tend to be inherited together, markers are often used as indirect ways of tracking the inheritance pattern of a gene that has not yet been identified, but whose approximate location is known. Thus, markers can assist breeders in developing populations of organism having a particular trait of interest. Gene-specific markers can be used to detect genetic variation among individuals which is more likely to affect phenotypes relating to the function of a specific gene. For example, variation in a gene- specific marker based on AaALDHI, rather than variation in an anonymous DNA marker, would be more likely linked to variation in content of artemisinin or related compounds, by virtue of its association with the relevant biosynthetic pathway. In one embodiment, a DNA marker for AaALDHI could be developed by sequencing the polymerase chain reaction amplified AaALDHI gene from a number of individual plants of Artemisia annua. Such sequencing would provide information about sequence polymorphisms within the gene. A range of methods available to those skilled in the art could be used to detect such polymorphisms, including cleaved amplified polymorphic sequences (CAPs) (Konieczy et al. 1993).

[0031 ] The presence of such gene-specific polymorphisms could be correlated with levels of artemisinin or related compounds and used in a breeding program to select and/or develop lines of Artemisia annua with enhanced levels of artemisinin or related compounds. That is, the variation in genetic structure may be detected in other plants, and the plants with the variation selectively bred to produce a population of plants having increased levels of dihydroartemisinic aldehyde, dihydroartemisinic acid, artemisinic acid and/or artemisinin compared to a population of control plants produced under similar conditions. Genetic markers are discussed in more detail in Bagge et al. 2007, Pfaff et al. 2003, Sandal et al. 2002 and Stone et al. 2002.

[0032] TILLING (Bagge et al. 2007, Comai et al. 2006, Henikoff, et al. 2004 and

Slade et al. 2005) involves treating seeds or individual cells with a mutagen to cause point mutations that are then discovered in genes of interest using a sensitive method for single-nucleotide mutation detection. Detection of desired mutations (e.g. mutations resulting in a change in expression of the gene product of interest) may be accomplished, for example, by PCR methods. For example, oligonucleotide primers derived from the gene (nucleic acid molecule) of interest, such as the nucleic acid molecules of the present invention, may be prepared and PCR may be used to amplify regions of the gene of interest from plants in the mutagenized population. Amplified mutant genes may be annealed to wild-type genes to find mismatches between the mutant genes and wild-type genes. Detected differences may be traced back to the plants which had the mutant gene thereby revealing which mutagenized plants will have the desired expression. These plants may then be selectively bred to produce a population having the desired expression.

[0033] Further features of the invention will be described or will become apparent in the course of the following detailed description. Brief Description of the Drawings

[0034] In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which:

[0035] Fig. 1 depicts the proposed biosynthetic pathway for artemisinin biosynthesis.

[0036] Fig. 2 depicts the nucleotide sequence (SEQ ID No.: 1 ) of the cDNA insert of pKT104 encoding Artemisia annua AaDBRL

[0037] Fig. 3 depicts the predicted amino acid sequence (SEQ ID No.: 2) of the protein encoded by Artemisia annua gene AaDBRL

[0038] Fig. 4 depicts the nucleotide sequence (SEQ ID No.: 3) of the open reading frame of the DNA insert in pKT032.

[0039] Fig. 5 depicts the predicted amino acid sequence (SEQ ID No.: 4) of the product of the AaDBRI insert in pKT032 in frame with an N-terminal His tag sequence.

[0040] Fig. 6 depicts the nucleotide sequence (SEQ ID No.: 5) of the cDNA insert of pKT150 encoding Artemisia annua AaALDHL

[0041] Fig. 7 depicts the predicted amino acid sequence (SEQ ID No.: 6) of the protein encoded by the Artemisia annua gene AaALDHL

[0042] Fig. 8 depicts the nucleotide sequence (SEQ ID No.: 7) of the open reading frame of the DNA insert in pKT041.

[0043] Fig. 9 depicts the predicted amino acid sequence (SEQ ID No.: 8) of the product of the AaALDHI insert in pKT041 in frame with an N-terminal His tag sequence.

[0044] Fig. 10 depicts a GC/MS of E. coli cell free extracts expressing AaDBRI tested with artemisinic aldehyde with (a) and without (b) added NADPH. The retention time and mass spectrum of the peaks at 14.94 and 15.03 min are equivalent to the standard artemisinic aldehyde (M₁ ⁺ 218) and ( 77S)-dihydroartemisinic aldehyde (M_r ⁺ 220), respectively.

[0045] Fig. 1 1 depicts a GC/MS of E. coli cell free extracts expressing AaALDHI tested with (77f?)-dihydroartemisinic aldehyde with (a) and without (b) added NADP. Diethyl ether extracts were analyzed as diazomethane derivatives. The retention time and mass spectrum of the peak at 15.8 min is equivalent to the standard diazomethane derivative of (77fi)-dihydroartemisinic acid (M⁺ 250).

Description of Preferred Embodiments

[0046] Materials and Methods:

[0047] Artemisinic aldehyde

[0048] Artemisinic acid was isolated from dichloromethane extracts of A. annua flower buds and leaves and was used to synthesize artemisinic aldehyde according to the method described by Chang et al. 2000, the disclosure of which is incorporated herein by reference.

[0049] Dihydroartemisinic acid

[0050] Dihydroartemisinic acid was isolated and purified from A. annua leaf material obtained from a "line 2/39" containing relatively high levels of the dihydroartemisinic acid using the method described for artemisinic acid in Teoh et al. 2006, the disclosure of which is incorporated herein by reference.

[0051] Dihydroartemisinic aldehyde

[0052] Dihydroartemisinic aldehyde was synthesized from the isolated dihydroartemisinic acid. The acid was converted to methyl dihydroartemisinate with excess diazomethane in diethyl ether at 0⁰C for 5 minutes. The ether and diazomethane were removed under a stream of nitrogen and the methyl ester was reduced to (11 R)- dihydroartemisinic alcohol with excess 1.5 M diisobutyl aluminum hydride in toluene at room temperature for 10 min under nitrogen. With subsequent extraction, oxidation to the aldehyde with pyridinium chlorochromate (Corey & Suggs 1975) and purification by HPLC the ( 77R)-dihydroartemisinic aldehyde was produced at an overall yield of 48% with >99% purity according to GC analysis.

[0053] Plant materials

[0054] Artemisia annua L. seeds were obtained from Elixir Farm Botanicals,

Brixey, Missouri, USA and from Pedro MeIiIIo de Magalhaes, State University of Campinas, Brazil (line 2/39). Seeds were germinated and grown in soil in a controlled environment chamber with 16 hour/25°C days and 8 hour/20°C nights. Plants that had reached the height of approximately 1.2 m (about 3 months) were transferred to flowering chamber with 12 hour/25°C days and 12 hour/20°C nights. Flower buds that developed after 19-21 days in the flowering chamber were harvested for total RNA isolation.

[0055] cDNA library construction and expressed sequence tag (EST) analysis

[0056] Total RNA was extracted and isolated from glandular trichomes and flower buds using a modified method described by Logeman, et al. 1987. cDNA synthesis from 1.5 micrograms of total RNA and construction of the trichome and flower bud cDNA library were carried out with Creator™ SMART™ cDNA Library Construction Kit (Clontech). A total of 6,239 clones and 2,208 clones for trichome and flower bud libraries, respectively were randomly picked and their DNA sequences determined. Sequencing was performed on an AB13700 DNA sequencer using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and the M13 reverse primer. DNA sequence traces were interpreted and vector and low quality sequences were eliminated using PHRED (Ewing et al. 1998) and LUCY (Chou & Holmes 2001). Clustering of the resulting EST dataset was done using STACKPACK (Miller et al. 1999) and sequence similarity was identified by BLAST (Altschul et al. 1990).

[0057] Isolation of full-length AaDBR 1 cDNA

[0058] The open reading frame (ORF) of a double bond reductase designated

AaDBRI , encoded by the EST clone pKT104, was obtained through PCR using gene- specific primers 5¹-CACCATGGAACAGCAACAAGAAG-3¹ (SEQ ID No.: 9) and 5'- TCATTCATGCGCAACCACCACCA-3' (SEQ ID No.: 10) and Vent polymerase (New England BioLabs, Cambridge, MA, USA). The resulting PCR product was cloned via the Gateway entry vector pENTR/D/TOPO (Invitrogen) into a Gateway destination vector, pDEST17 (Invitrogen) to generate a bacteria expression clone pKT032. The ORF of AaDBRI was cloned in frame with the 6X His-tag (SEQ ID No.: 13) at the N-terminal of AaDBRL

[0059] Isolation of full-length AaALDHI cDNA

[0060] The open reading frame (ORF) of an aldehyde dehydrogenase designated

AaALDHI ₁ encoded by the EST clone pKT150, was obtained through PCR using gene- specific primers 5'-CACCATGAGCTCAGGAGCTAAT-3' (SEQ ID No.:11 ) and δ'-TTAAAGCCACGGGGAATCATAT-S' (SEQ ID NO. 12) and Vent polymerase (New England BioLabs, Cambridge, MA, USA). The resulting PCR product was cloned via the Gateway entry vector pENTR/D/TOPO (Invitrogen) into a Gateway destination vector, pDEST17 (Invitrogen) to generate a bacterial expression clone pKT041. The ORF of AaALDHI was cloned in frame with the 6X His-tag (SEQ ID No.: 13) at the N-terminal of AaALDHL

[0061] Expression in E. coli

[0062] The plasmid pKT032 or pKT041 was introduced into E. coli strain

BL21(DE3) (Novagen) using heat shock at 42⁰C. The GUS gene (Invitrogen) was cloned into pDEST17 to replace the ccdB gene and the construct pDEST-GUS introduced into the E. coli strain BL21(DE3) was used as a control. Transformants were grown on Luria Broth (LB) and selected on ampicillin (100 μg/mL) at 37⁰C for 24 hours. A single colony containing pKT032 or pKT041 was used to inoculate 5 mL of LB liquid medium with ampicillin (LBA) and grown at 37⁰C overnight with shaking. The overnight culture was used to inoculate 250 mL of LBA liquid medium and grown at 37⁰C with shaking to an OD₆₀₀ of 0.6 per mL followed by induction with 1 mM IPTG and grown at 3O⁰C overnight with shaking. Cells were pelleted at 2,00Og at 4⁰C for 10 minutes. The pelleted cells were resuspended in 6 mL of lysis buffer consisting of 50 mM sodium phosphate, pH 8.0, 0.1 M NaCI, 20 mM imidazole and 1 mM phenylmethylsufonyl fluoride (PMSF). Cells were lysed with lysozyme (0.2 mg/mL of cells) on ice for 30 minutes followed by sonication on ice with 30s pulse (5X). Protein concentration was determined by Bradford assay (Bio-Rad). The AaDBRI protein was detected by silver stain on SDS gel and confirmed by Western Blot using Anti-His antibody (Invitrogen). The AaALDHI protein was detected by Rapid Stain (Bioscience, St. Louis, MO) on SDS gel.

[0063] Purification of recombinant AaALDHI

[0064] Cell-free extract of recombinant AaALDHI was prepared as described above. The cell-free extract was centrifuged at 20,000 g at 4⁰C for 15 minutes to remove any remaining insoluble materials before loading onto a His-Trap FF column (Amersham Bioscience, NJ) equilibrated with binding buffer (20 mM sodium phosphate buffer containing 500 mM NaCI and 20 mM imidazole at pH 7.5). The column was washed with 5 column volume of binding buffer and the recombinant AaALDHI eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCI, pH 7.5) containing increasing concentration of imidazole in a step-wise fashion. The eluted fractions were concentrated and desalted in centrifugal filter devices (Amicon Ultra - 15) (Millipore, MA) following manufacturer's protocol. The purity of the recombinant AaALDHI was checked with SDS gel stained with Rapid Stain (Biosciences, St. Louis, MO). [0065] In Vitro Cell-free Assays

[0066] Cell-free extracts of recombinant His-tagged AaDBRI protein were assayed with artemisinic aldehyde, followed by analysis by gas chromatography/mass spectrometry. Enzyme reactions were initiated by adding the substrate (5 μg) to 500 μL sodium phosphate buffer (50 mM, pH 7.5) containing 10% sorbitol, 1 mM NADPH, 2 mM DTT and 0.8 μg of enzyme. Negative controls were carried out with boiled proteins, without NADPH and with extracts from E. coli into which the construct pDEST17-GUS had been introduced. Reactions were allowed to proceed for 30 minutes at 30°C with shaking and immediately stopped by extracting twice with 700 μL diethyl ether. The ether extracts were pooled, evaporated and taken up in 20 μL ethyl acetate (Sigma) followed by GC-MS analysis.

[0067] Cell-free extract of recombinant His-tagged AaALDHI protein were assayed with dihydroartemisinic aldehyde and other substrates, followed by analysis by gas chromatography/mass spectrometry. Enzyme reactions were initiated by adding the substrate (5 μg) to 500 μL Tris-HCI buffer (50 mM, pH 8.5) containing, 1 mM NADP, and 1.0 μg of enzyme. Negative controls were carried out with boiled proteins, without NADP and with extracts from E coli into which the construct pDEST17-GUS had been introduced. Reactions were allowed to proceed for 30 minutes at 3O⁰C with shaking and immediately stopped by extracting twice with 700 μL diethyl ether. The ether extracts were pooled, derivatized with diazomethane, evaporated and taken up in 20 μL dichloromethane (Sigma) followed by GC-MS analysis.

[0068] Characterization of purified recombinant AaALDHI

[0069] The linearity of the assay with respect to time and protein concentration was first established and the operational saturation of substrate and cofactor determined. The pH optimum was determined by the standard assay in 50 mM buffer (sodium phosphate, Tris-HCI and CHES) from pH 6.0 to 10.0 at 0.5 unit interval containing 1 mM NADP and 1.5 micrograms of the purified recombinant AaALDHL Kinetic parameters were determined in 50 mM Tris-HCI buffer, pH 8.5 by varying the concentration of the substrates. Kinetic constants were determined by non-linear regression analysis using GraphPad software (GraphPad Software Inc. San Diego, CA) and the results presented are the means of three independent experiments. Substrates specificity was determined at optimum reaction conditions with substrates concentration at 10 times the estimated Km value. Substrates tested include artemisinic aldehyde, (77f?)-dihydroartemisinic aldehyde, artemisinic alcohol, dihydroartemisinic alcohol, octanal, nonanal, 2-phenyl propionaldehyde, 3-cyclohexyl propionaldehyde, 2-hexen-1-al, syringaldehyde.

[0070] Results:

[0071] Expressed sequence tags (sequences of randomly picked cDNA clones) were generated from developing trichomes and flower buds of Artemisia annua and analyzed.

[0072] cDNA clones with sequences similar to monoterpene double-bond reductases were resequenced and these sequences were assembled. These were deemed to be derived from a single Artemisia annua gene which was called AaDBRL The consensus nucleotide sequence of the AaDBRI mRNA is shown in Fig. 2 (SEQ ID No.: 1 ). The corresponding amino acid sequence is shown in Fig. 3 (SEQ ID No.: 2).

[0073] cDNA clones with sequences similar to aldehyde dehydrogenase were resequenced and these sequences were assembled. These were deemed to be derived from a single Artemisia annua gene which was called AaALDHL The consensus nucleotide sequence of the AaALDHI mRNA is shown in Fig. 6 (SEQ ID No.: 5). The corresponding amino acid sequence is shown in Fig. 7 (SEQ ID No.: 6).

[0074] For initial functional studies of AaDBRI , an RT-PCR product was prepared and cloned into E. coli expression vector pDEST17 to give the clone pKT032. The nucleotide sequence of the open reading frame of the DNA insert of pKT032 is given in Fig. 4 (SEQ ID No.: 3) and the corresponding protein product including the N-terminal His tag fusion is given in Fig. 5 (SEQ ID No.: 4). The plasmid pKT032 was introduced into the E. coli (DE3) strain (Novagen) and cell-free extracts was assayed with various isoprenoid substrates followed by analysis by gas chromatography/mass spectrometry. Fig. 10 shows the results of this analysis indicating the NADPH-dependent formation of ( 11 S)- dihydroartemisinic aldehyde as the major product. In a separate experiment, extracts from E. coli into which pKT032 had not been introduced did not support the production of dihydroartemisinic aldehyde in the presence of NADPH. It is predicted that the wild type product of AaDBRI will have similar artemisinic aldehyde double bond reductase activity as the His tag fusion protein product of pKT032.

[0075] For initial functional studies of AaALDHI , a PCR product was prepared and cloned into E. coli expression vector pDEST17 to give the clone pKT041. The nucleotide sequence of the open reading frame of the DNA insert of pKT041 is given in Fig. 8 (SEQ ID No.: 7) and the corresponding protein product including the N-terminal His tag fusion is given in Fig. 9 (SEQ ID No.: 8). The plasmid pKT041 was introduced into the E. coli (DE3) strain (Novagen) and cell-free extracts was assayed with ( 11 R)- dihydroartemisinic aldehyde followed by analysis by gas chromatography/mass spectrometry. The recombinant AaALDHI protein was purified from the cell-free extract and its kinetic parameters were determined. The purified recombinant AaALDHI protein functions best at pH 8.5. The recombinant protein was tested with different substrates (see Materials and Methods) at the optimum assay conditions. Artemisinic aldehyde besides (77R)-dihydroartemisinic aldehyde was found to be a substrate for the recombinant AaALDHL The K_m and V_max values determined for dihydroartemisinic acid were 8.79 μM and 143.8 pkat/μg protein, respectively and for artemisinic aldehyde the K_m and V_max were 2.62 μM and 28.6 pkat/μg protein, respectively. Fig. 11 shows the results of the analysis for (77f?)-dihydroartemisinic aldehyde indicating the NADP-dependent formation of dihydroartemisinic acid. In separate experiment, extracts from E. coli into which pKT041 had not been introduced did not support the production of dihydroartemisinic acid in the presence of NADP. It is predicted that the wild type product of AaALDHI will have similar artemisinic/dihydroartemisinic aldehyde dehydrogenase activity as the His-tag fusion protein product of pKT041.

[0076] References: The disclosures of the following references are incorporated herein by reference in their entirety.

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[0077] Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.

Claims

Claims:

1. Isolated nucleic acid molecule comprising a nucleotide sequence having at least 70% nucleotide sequence identity to SEQ ID No.: 3 and encoding an artemisinic aldehyde double bond reductase.

2. Isolated nucleic acid molecule comprising a nucleotide sequence encoding an artemisinic aldehyde double bond reductase having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 2.

3. The isolated nucleic acid molecule of claim 1 or 2 having a nucleotide sequence of SEQ ID No.: 1.

4. Isolated nucleic acid molecule comprising a nucleotide sequence having at least 70% nucleotide sequence identity to SEQ ID No.: 7 and encoding an artemisinic/dihydroartemisinic aldehyde dehydrogenase.

5. Isolated nucleic acid molecule comprising a nucleotide sequence encoding an artemisinic/dihydroartemisinic aldehyde dehydrogenase having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 6.

6. The isolated nucleic acid molecule of claim 4 or 5 having a nucleotide sequence of SEQ ID No.: 5.

7. The isolated nucleic acid molecule of any one of claims 1 to 6 derived from Artemisia annua.

8. Purified artemisinic aldehyde double bond reductase having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 2.

9. The reductase of claim 8 having a nucleotide sequence of SEQ ID No.: 2.

10. Purified artemisinic/dihydroartemisinic aldehyde dehydrogenase having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 6.

11. The dehydrogenase of claim 10 having a nucleotide sequence of SEQ ID No.: 6.

12. Use of one or more isolated nucleic acid molecules of any one of claims 1 to 7 in the production of dihydroartemisinic aldehyde, dihydroartemisinic acid and/or artemisinic acid.

13. Use of one or more of a purified artemisinic aldehyde double bond reductase or artemisinic/dihydroartemisinic aldehyde dehydrogenase encoded by one or more isolated nucleic acid molecules of any one of claims 1 to 7 in the production of dihydroartemisinic aldehyde, dihydroartemisinic acid and/or artemisinic acid.

14. Process for producing dihydroartemisinic aldehyde, dihydroartemisinic acid and/or artemisinic acid comprising expressing or overexpressing one or more isolated nucleic acid molecules of any one of claims 1 to 7 in a host cell.

15. The process of claim 14, further comprising expressing or overexpressing in the host cell one or more nucleic acid molecules encoding amorpha-4,11-diene synthase and/or amorpha-4,11-diene hydroxylase.

16. Process for producing dihydroartemisinic aldehyde, dihydroartemisinic acid and/or artemisinic acid comprising producing or overproducing a protein having an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 2 and/or an amino acid sequence with at least 70% amino acid sequence identity to SEQ ID No.: 6 in a host cell.

17. The process of any one of claims 14 to 16, wherein the host cell is a plant cell, a yeast cell or a bacterial cell.

18. A method of selecting or developing plants with altered dihydroartemisinic aldehyde, dihydroartemisinic acid, artemisinic acid and/or artemisinin levels in a population of plants that naturally produces dihydroartemisinic aldehyde, dihydroartemisinic acid, artemisinic acid and/or artemisinin comprising:

detecting a target plant having altered levels of dihydroartemisinic aldehyde, dihydroartemisinic acid, artemisinic acid and/or artemisinin compared to a control plant provided under similar conditions;

isolating at least a portion of an artemisinic aldehyde double bond reductase gene or artemisinic/dihydroartemisinic aldehyde dehydrogenase gene of the target plant and comparing the nucleotide sequence of the at least a portion to SEQ ID No. 1 , SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7 to detect a variation from SEQ ID No. 1 , SEQ ID No. 3, SEQ ID No. 5 or SEQ ID No. 7;

detecting the variation in other plants; selectively breeding the plants with the variation to produce a population of plants having altered levels of dihydroartemisinic aldehyde, dihydroartemisinic acid, artemisinic acid and/or artemisinin compared to a population of control plants produced under similar conditions.

19. A method of altering dihydroartemisinic aldehyde, dihydroartemisinic acid, artemisinic acid and/or artemisinin levels in a population of plants that naturally produces dihydroartemisinic aldehyde, dihydroartemisinic acid and/or artemisinic acid comprising:

providing a population of mutated plants;

detecting a target mutated plant within the population of mutated plants, the target mutated plant having an altered expression of an artemisinic aldehyde double bond reductase gene or an artemisinic/dihydroartemisinic aldehyde dehydrogenase gene or altered activity of an artemisinic aldehyde double bond reductase enzyme or an artemisinic/dihydroartemisinic aldehyde dehydrogenase enzyme compared to a control plant provided under similar conditions,

said detecting comprising using primers developed from a nucleic acid molecule as defined in any one of claims 1 to 7 to PCR amplify regions of the artemisinic aldehyde double bond reductase gene or artemisinic/dihydroartemisinic aldehyde dehydrogenase gene from mutated plants in the population of mutated plants, identifying mismatches between the amplified regions and corresponding regions in wild-type gene that lead to the altered expression or altered activity, and identifying the mutated plant that contains the mismatches; and,

selectively breeding the target mutated plant to produce a population of plants having altered expression of artemisinic aldehyde double bond reductase gene or artemisinic/dihydroartemisinic aldehyde dehydrogenase gene or altered activity of artemisinic aldehyde double bond reductase enzyme or artemisinic/dihydroartemisinic aldehyde dehydrogenase enzyme compared to a population of control plants produced under similar conditions.