AU780594B2 - Cytochrome P450 oxygenases and their uses - Google Patents

Description

24/01 2005 15:51 FAX 61 3 92438333 GRIFFITH HACK 4IPAUST'RALIA [a 006 -1- CYTOCHROME P450 OXYGENASES AND THEIR USES FIELD OF THE INVENTION The invention relates to oxygenase enzymes and methods of using such enzymes to produce Taxol (paclitaxel) and related taxoids.

INTRODUCTION

Cytochrome P450 Cytochrome P450 proteins are enzymes that have a unique sulfur atom ligated to the heme iron and that, when reduced, form carbon monoxide complexes.

When complexed to carbon monoxide they display a major absorption peak (Soret band) near 450 nm. There are numerous members of the cytochrome P450 group including enzymes from both plants and animals. Members of the cytochrome P450 group can catalyse reactions such as unspecific monooxygenation, 15 monooxygenation, steroid 1 f1-monooxygenation, and cholesterol monooxygenation (Smith et al. Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, New York, 1997).

Paclitaxel The complex diterpenoid Taxol Bristol-Myers Squibb; common name paclitaxel) (Wani et al., J. Am. Chem. Soc. 93 2325-2327, 1971) is a potent antimitotic agent with excellent activity against a wide range of cancers, including ovarian and breast cancer (Arbuck and Blaylock, Taxol: Science and Applications, CRC Press, Boca Raton, 397-415, 1995 Holmes et al., ACS Symposium Series 583:31-57, 1995). Taxol 25 was isolated originally from the bark of the Pacific ycw (Taxus brevifolia). For a number of years, Taxol was obtained exclusively from yew bark, but low yields of this compound from the natural source coupled to the destructive nature of the harvest, prompted new methods of Taxol production to be developed. Taxol currently is produced primarily by semisynthesis from advanced R \r-bCcca\kccp\cpecifiCatlonl\14887-01 amondsnt.duc 24/01/05 COMS ID No: SBMI-01090146 Received by IP Australia: Time 16:00 Date 2005-01-24 WO 01/34780 PCT/US00/31254 taxane metabolites (Holton et al., Taxol: Science and Applications, CRC Press, Boca Raton, 97-121, 1995) that are present in the needles (a renewable resource) of various Taxus species. However, because of the increasing demand for this drug both for use earlier in the course of cancer intervention and for new therapeutic applications (Goldspiel, Pharmacotherapy 17:110S-125S, 1997), availability and cost remain important issues. Total chemical synthesis of Taxol currently is not economically feasible. Hence, biological production of the drug and its immediate precursors will remain the method of choice for the foreseeable future. Such biological production may rely upon either intact Taxus plants, Taxus cell cultures o0 (Ketchum et al., Biotechnol. Bioeng. 62:97-105, 1999), or, potentially, microbial systems (Stierle et al., J. Nat. Prod. 58:1315-1324, 1995). In all cases, improving the biological production yields of Taxol depends upon a detailed understanding of the biosynthetic pathway, the enzymes catalyzing the sequence of reactions, especially the rate-limiting steps, and the genes encoding these proteins. Isolation of genes encoding enzymes involved in the pathway is a particularly important goal, since overexpression of these genes in a producing organism can be expected to markedly improve yields of the drug.

The Taxol biosynthetic pathway is considered to involve more than 12 distinct steps (Floss and Mocek, Taxol: Science and Applications, CRC Press, Boca Raton, 191-208, 1995; and Croteau et al., Curr. Top. Plant Physiol: 15:94-104, 1996). However, very few of the enzymatic reactions and intermediates of this complex pathway have been defined. The first committed enzyme of the Taxol pathway is taxadiene synthase (Koepp et al., J. Biol. Chem. 270:8686-8690, 1995) that cyclizes the common precursor geranylgeranyl diphosphate (Hefner et al., Arch.

Biochem. Biophys. 360:62-74, 1998) to taxadiene (Fig. The cyclized intermediate subsequently undergoes modification involving at least eight oxygenation steps, a formal dehydrogenation, an epoxide rearrangement to an oxetane, and several acylations (Floss and Mocek, Taxol: Science and Applications, CRC Press, Boca Raton, 191-208, 1995; and Croteau et al., Curr. Top. Plant Physiol. 15:94-104, 1996). Taxadiene synthase has been isolated from T. brevifolia and characterized (Hezari et al., Arch. Biochem. Biophys. 322:437-444, 1995), the mechanism of action defined (Lin et al., Biochemistry 35:2968-2977, 1996), and the WO 01/34780 PCT/US00/31254 corresponding cDNA clone isolated and expressed (Wildung and Croteau, J. Biol.

Chem. 271:9201-9204, 1996).

The second specific step of Taxol biosynthesis is an oxygenation (hydroxylation) reaction catalyzed by taxadiene-5a-hydroxylase. The enzyme has been demonstrated in Taxus microsome preparations (Hefner et al., Methods Enzymol. 272:243-250, 1996), shown to catalyze the stereospecific hydroxylation of taxa-4(5),l1(12)-diene to taxa-4(20), 1l(12)-dien-5a-ol with double-bond rearrangement), and characterized as a cytochrome P450 oxygenase (Hefner et al., Chemistry and Biology 3:479-489, 1996).

Since the first specific oxygenation step of the Taxol pathway was catalyzed by a cytochrome P450 oxygenase, it was logical to assume that subsequent oxygenation (hydroxylation and epoxidation) reactions of the pathway would be carried out by similar cytochrome P450 enzymes. Microsomal preparations (Hefner et al., Methods Enzymol. 272:243-250, 1996) were optimized for this purpose, and shown to catalyze the hydroxylation of taxadiene or taxadien-5o-ol to the level of a pentaol (see Fig. 2 for tentative biosynthetic sequence and structures based on the evaluation oftaxane metabolite abundances (Croteau et al., Curr. Topics Plant Physiol. 15:94-104, 1995)), providing evidence for the involvement of at least five distinct cytochrome P450 taxane (taxoid) hydroxylases in this early part of the pathway (Hezari et al., Planta Med. 63:291-295, 1997).

Also, the remaining three oxygenation steps (Cl and C7 hydroxylations and an epoxidation at C4-C20; see Figs. 1 and 3) likely are catalyzed by cytochrome P450 enzymes, but these reactions reside too far down the pathway to observe in microsomes by current experimental methods (Croteau et al., Curr. Topics Plant Physiol. 15:94-104, 1995; and Hezari et al., Planta Med. 63:291-295, 1997). Since Taxus (yew) plants and cells do not appear to accumulate taxoid metabolites bearing fewer than six oxygen atoms hexaol or epoxypentaol) (Kingston et al., Prog.

Chem. Org. Nat. Prod. 61:1-206, 1993), such intermediates must be rapidly transformed down the pathway, indicating that the oxygenations (hydroxylations) are relatively slow pathway steps and, thus, important targets for gene cloning.

24/01 2005 15:51 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRALIA Q0077 4 Isolation of the genes encoding the oxygenases that catalyze the oxygcnase steps of Taxol biosynthesis would represent an important advance in efforts to increase Taxol and taxoid yields by genetic engineering and in vitro synthesis.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

In the claims which follow and in the description of the invention, except 15 where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

SUMMARY OF THE INVENTION S. The invention stems from the discovery of twenty-one amplicons S* (regions of DNA amplified by a pair of primers using the polymerase chain reaction These amplicons can be used to identify oxygenases, for example, the oxygenases shown in SEQ ID NOS: 56-68 and 87-92 that are encoded by the nucleic acid sequences shown in SEQ ID NOS: 43-55 and 81-86. These sequences are isolated from the Taxus genus, and the respective oxygenases are useful for the synthetic production of Taxol and related taxoids, as well as intermediates within the Taxol biosynthetic pathway, and other taxoid derivatives. The sequences also can be used for the creation of transgenic organisms that either produce the oxygenases for subsequent in vitro use, or produce the oxygenases in vivo so as to alter the level of Taxol and taxoid production within the transgenic organism.

Another aspect of the invention provides the nucleic acid sequences shown in SEQ ID NOS: 1-21 and the corresponding amino acid sequences shown in SEQ ID NOS: 22-42, respectively, as well as fragments of these nucleic acid sequences and amino acid sequences. These sequences are useful for isolating the nucleic acid and amino acid sequences corresponding to full-length oxygenases. These amino acid sequences and nucleic acid sequences are also useful for creating specific binding H.\rhbccao\keep\peciftictLons\14887-OL amendment .doc 23/01/05 COMS ID No: SBMI-01090146 Received by IP Australia: Time 16:00 Date 2005-01-24 24/01 2005 15:51 FAX 61 3 92438333 GRIFFITH HACK 4IPAUSTRALIA IZj008 4a agents that recognize the corresponding oxygenases.

Accordingly, another aspect of the invention provides for the identification of oxygenases. and fragments of oxygenases that have amino acid and nucleic acid sequences that vary from the disclosed sequences. For example, the invention provides oxygenase amaino acid sequences that vary by one or more conservative amiuno acid substitutions, or that share at least 50% sequence identity with the amino acid sequences provided while maintaining oxygenase activity.

The nucleic acid sequences encoding the oxygenases and fragments of the oxygenases that maintain taxoid oxygenase and/or CO binding activity can be cloned, using standard molecular biology techniques, into vectors. These vectors UB \rebeccam\kccp.Pcification~a\l4I67-01 auwnduenta dOC 24/01/0S COMS ID No: SBMI-01090146 Received by IP Australia: Time 16:00 Date 2005-01-24 WO 01/34780 PCT/US0/31254 then can be used to transform host cells. Thus, a host cell can be modified to express either increased levels of oxygenase or decreased levels of oxygenase.

Another aspect of the invention provides methods for isolating nucleic acid sequences encoding full-length oxygenases. The methods involve hybridizing at least ten contiguous nucleotides of any of the nucleic acid sequences shown in SEQ ID NOS: 1-21, 43-55, and 81-86 to a second nucleic acid sequence, wherein the second nucleic acid sequence encodes a taxoid oxygenase and/or maintains CO binding activity. This method can be practiced in the context of, for example, Northern blots, Southern blots, and the polymerase chain reaction (PCR). Hence, the invention also provides the oxygenases identified by this method.

Yet another aspect of the invention involves methods of adding at least one oxygen atom to at least one taxoid. These methods can be practiced in vivo or in vitro, and can be used to add oxygen atoms to various intermediates in the Taxol biosynthetic pathway, as well as to add oxygen atoms to related taxoids that are not necessarily on a Taxol biosynthetic pathway. These methods include for example, adding oxygen atoms to acylation or glycosylation variants of paclitaxel, baccatin III, or 10-deacetyl-baccatin III. Such variants include, cephalomannine, xylosyl paclitaxel, 10-deactyl paclitaxel, paclitaxel C, 7-xylosyl baccatin III, 2-debenzoyl baccatin III, 7-xylosyl 10-baccatin III and 2-debenzoyl 10-baccatin III.

Yet another aspect of the invention involves methods of contacting the reduced form of any one of the disclosed oxygenases with carbon monoxide and detecting the carbon monoxide/oxygenase complex.

SEQUENCE LISTINGS The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.

SEQ ID NOS: 1-21 are the nucleic acid sequences of the 21 different respective amplicons generated from the mRNA-reverse transcription-PCR.

WO 01/34780 PCT/US00/31254 SEQ ID NOS: 22-42 are the deduced amino acid sequences of the nucleic acid sequences shown in SEQ ID NOS: 1-21, respectively.

SEQ ID NOS: 43-55 are the full-length nucleic acid sequences of 13 respective oxygenases.

SEQ ID NOS: 56-68 are the deduced amino acid sequences of the nucleic acid sequences shown in SEQ ID NOS: 43-55, respectively.

SEQ ID NOS: 69-72 are the PCR primers used in the RACE protocol.

SEQ ID NOS: 73-80 are PCR primers used to amplify the 21 different amplicons.

SEQ ID NOS: 81-86 are the full-length nucleic acid sequences of 6 respective oxygenases.

SEQ ID NOS: 87-92 are the full-length amino acid sequences of 6 respective oxygenases corresponding to the nucleic acid sequences show in SEQ ID NOS: 81-90, respectively.

SEQ ID NOS: 93 and 94 are PCR primers that were used to clone oxygenases into FastBac-1 vector (Life Technologies).

FIGURES

Fig. I shows an outline of early steps of the Taxol biosynthetic pathway illustrating cyclization of geranylgeranyl diphosphate to taxadiene by taxadiene synthase hydroxylation and rearrangement of the parent olefin to by taxadiene 5a-hydroxylase acetylation by taxadienol-O-acetyl transferase and hydroxylation to taxadien-5a-acetoxy-10-ol by the taxane The broken arrow indicates several as yet undefined steps.

WO 01/34780 PCT/USOO/31254 Fig. 2 shows the proposed sequence for the hydroxylation of taxa- 1(12)-diene to the level of a pentaol based on the relative abundances of naturally occurring taxoids. The reactions are catalyzed by cytochrome P450 oxygenases.

Fig. 3 shows a possible mechanism for the construction of the oxetane ring of Taxol from the 4(20)-ene-5a0-acetoxy functional grouping. Cytochrome P450-catalyzed epoxidation of the 4(20)-double bond, followed by intramolecular acetate migration and oxirane ring opening, could furnish the oxetane moiety.

Fig. 4 shows P450-specific forward primers that were used for differential display of mRNA-reverse transcription-polymerase chain reaction (DD- RT-PCR). Eight nondegenerate primers were necessary to cover all possible nucleotide sequences coding for the proline, phenylalanine, glycine (PFG) motif.

Anchors were designed by Clontech as components of the kit.

Figs. 5A and 5D show the relationship between the full-length amino acid sequences of the isolated oxygenases. Fig. 5A is a dendrogram showing peptide sequence relationships between some published, related plant cytochrome P450s and those cloned from T. cuspidata. For the published sequences, the first four letters of each name are genus and species abbreviations, CYP is the abbreviation for cytochrome P450, the following two numbers indicate the P450 family, and any additional letters and numbers refer to the subfamily. Cloned sequences from T. cuspidata are denoted by followed by a number. The genus and species abbreviations are as follows: Lius Linum usitatissimum; Paar Parthenium argentatum; Caro Catharanthus roseus; Some Solanum melongena; Arth Arabidopsis thaliana; Hetu Helianthus tuberosus; Ziel Zinnia elegans; Poki Populus kitamkensis; Glma Glycine max; Phau Phaseolus aureus; Glee Glycyrrhiza echinata; Mesa Medicago saliva; Pisa Pisum sativum; Peer Petroselinum crispum; Zema Zea mays; Nita Nicotiana tabacum; Eugr Eustoma grandiflorum; Getr Gentiana triflora; Peam Persea americana; Mepi WO 01/34780 PCT/USOO/31254 Menthapiperita; Thar Thlaspi arvense; Best Berberis stolonifera; Soly Solanum lycopersicum; Sobi Sorghum bicolor; Potr Populus tremuloides; Soch Solanum chacoense; Nera Nepeta racemosa; Came Campanula medium; Pehy Petunia hybrida. Fig. 5B shows a pairwise comparison of certain Taxus cytochrome P450 clones. Fig. 5C is a dendrogram showing the relationships between the fulllength peptide sequences of the disclosed proteins. The dendrogram was created using the Clustral Method. The sequence identity data used as the basis of the dendrogram was created using the Sequence Distance function of the Megalign program of the lasergene (Version 99) package from DNAStar T M Fig. 5D is a similarity/identity table. The sequence identity data was generated using the same program as that used for generating the dendrogram shown in Fig. 5C and the similarity data was generated using the Olddistance function of GCG M (version GCG1O).

Figs. 6A-6E show a reversed-phase HPLC radio-trace illustrating the conversion of [20- 3

H

2 ]taxa-4(20), l(12)-dien-5a-ol to more polar products by yeast transformants expressing Taxus cuspidata P450 genes and mass spectrum results.

Fig. 6A shows the HPLC radio-trace of the authentic substrate [20- 3

H

2 ]taxa- 4(20),11(12)-dien-5a-ol. Figs. 6B and 6C show the HPLC radio-trace of the substrate [20- 3

H

2 ]taxa-4(20),l 1(12)-dien-5a-ol (26.33 min) and more polar products (retention ~15 min) obtained after incubation with yeast transformed with clones F12 (SEQ ID NO: 43) and F9 (SEQ ID NO: 48), respectively. Figs. 6D and 6E show the mass spectrum of the products (at 15.76 minutes and at 15.32 minutes, respectively) formed during the incubation of taxadien-5a-ol with yeast transformants expressing clones F12 and F9, respectively. Cytochrome P450 clones F14 (SEQ ID NO: 51) and F51 (SEQ. ID NO: 47) behaved similarly in yielding diol products.

Fig. 7 shows a 500 MHz proton NMR spectrum of the taxadien-diol monoacetate in benzene-d 6 WO 01/34780 PCT/US00/31254 Fig. 8 shows a 'H detected two-dimensional heteronuclear single quantum coherence (HSQC) NMR spectrum of the unknown taxadien-diol monoacetate.

Figs. 9A and 9B show a two-dimensional homonuclear rotating frame NMR of the diol monoacetate. Fig. 9A is a total correlation spectrum (TOSCY) and Fig. 9B is a rotating frame n.O.e. (ROESY).

Figs. 10A-10E show slices from the TOCSY spectrum taken along the F2, directly detected, axis.

Figs. 11A-11E show slices from the ROESY spectrum taken along the F2, directly detected, axis.

DETAILED DESCRIPTION Explanations Host cell: A "host cell" is any cell that is capable of being transformed with a recombinant nucleic acid sequence. For example, bacterial cells, fungal cells, plant cells, insect cells, avian cells, mammalian cells, and amphibian cells.

Taxoid: A "taxoid" is a chemical based on the Taxane ring structure as described in Kingston et al., Progress in the Chemistry of Organic Natural Products, Springer-Verlag, 1993.

Isolated: An "isolated" biological component (such as a nucleic acid or protein or organelle) is a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, other chromosomal and extra-chromosomal DNA, RNA, proteins, and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

WO 01/34780 PCT/US00/31254 Orthologs: An "ortholog" is a gene encoding a protein that displays a function similar to a gene derived from a different species.

Homologs: "Homologs" are multiple nucleotide sequences that share a common ancestral sequence and that diverged when a species carrying that ancestral sequence split into at least two species.

Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified enzyme or nucleic acid preparation is one in which the subject protein or nucleotide, respectively, is at a higher concentration than the protein or nucleotide would be in its natural environment within an organism. For example, a preparation of an enzyme can be considered as purified if the enzyme content in the preparation represents at least 50% of the total protein content of the preparation.

Vector: A "vector" is a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences, such as an origin of replication, that permit the vector to replicate in a host cell. A vector may also include one or more screenable markers, selectable markers, or reporter genes and other genetic elements known in the art.

Transformed: A "transformed" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle-gun acceleration.

DNA construct: The term "DNA construct" is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA, or RNA origin.

The term "construct" is intended to indicate a nucleic acid segment that may be WO 01/34780 PCT/US00/31254 single- or double-stranded, and that may be based on a complete or partial naturally occurring nucleotide sequence encoding one or more of the oxygenase genes of the present invention. It is understood that such nucleotide sequences include intentionally manipulated nucleotide sequences, subjected to site-directed mutagenesis, and sequences that are degenerate as a result of the genetic code. All degenerate nucleotide sequences are included within the scope of the invention so long as the oxygenase encoded by the nucleotide sequence maintains oxygenase activity as described below.

Recombinant: A "recombinant" nucleic acid is one having a sequence that is not naturally occurring in the organism in which it is expressed, or has a sequence made by an artificial combination of two otherwise-separated, shorter sequences.

This artificial combination is accomplished often by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. "Recombinant" also is used to describe nucleic acid molecules that have been artificially manipulated, but contain the same control sequences and coding regions that are found in the organism from which the gene was isolated.

Specific binding agent: A "specific binding agent" is an agent that is capable of specifically binding to the oxygenases of the present invention, and may include polyclonal antibodies, monoclonal antibodies (including humanized monoclonal antibodies) and fragments of monoclonal antibodies such as Fab, F(ab') 2 and Fv fragments, as well as any other agent capable of specifically binding to the epitopes on the proteins.

cDNA (complementary DNA): A "cDNA" is a piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.

WO 01/34780 PCT/US00/31254 ORF (open reading frame): An "ORF" is a series of nucleotide triplets (codons) coding for amino acids without any termination codons. These sequences are usually translatable into respective polypeptides.

Operably linked: A first nucleic acid sequence is "operably linked" with a second nucleic acid sequence whenever the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Probes and primers: Nucleic acid probes and primers may readily be prepared based on the amino acid sequences and nucleic acid sequences provided by this invention. A "probe" comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. Probes are typically shorter in length than the sequences from which they are derived cDNA or gene sequences). For example, the amplicons shown in SEQ ID NOS: 1-21 and fragments thereof can be used as probes. One of ordinary skill in the art will appreciate that probe specificity increases with the length of the probe. For example, a probe can contain less than 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 100 bp, or 50 bp of constitutive bases of any of the oxygenase encoding sequences disclosed herein.

Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed, in Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987.

"Primers" are short nucleic acids, preferably DNA oligonucleotides nucleotides or more in length. A primer may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and WO 01/34780 PCT/US00/31254 the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, by the polymerase chain reaction (PCR), or other nucleic-acid amplification methods known in the art.

Methods for preparing and using probes and primers are described, for example, in references such as Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987; and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA). One of skill in the art will appreciate that the specificity of a particular probe or primer increases with the length of the probe or primer. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with higher specificity than a corresponding primer of only nucleotides in length. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise, for example, 10, 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.

Sequence identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.

Methods for aligning sequences for comparison are well known in the art.

Various programs and alignment algorithms are described in: Smith Waterman, Adv. Appl. Math. 2:482, 1981; Needleman Wunsch, J. Mol. Biol. 48:443, 1970; Pearson Lipman, Proc. Natl. Acad Sci. USA 85:2444, 1988; Higgins Sharp, Gene 73:237-244, 1988; Higgins Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890,.1988; Huang, et al., Computer Applications WO 01/34780 PCT/US00/31254 in the Biosciences 8:155-165, 1992; and Pearson et al., Methods in Molecular Biology 24:307-331, 1994. Altschul et al., J. Mol. Biol. 215:403-410, 1990, presents a detailed consideration of sequence-alignment methods and homology calculations.

The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST

T

Altschul et al.. J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Intemet, for use in connection with the sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the internet under the help section for BLASTT

M

For comparisons of amino acid sequences of greater than about 30 amino acids, the "Blast 2 sequences" function of the BLASTT M program is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 45%, at least 50%, at least 60%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity.

As mentioned above, 'Sequence identity' can be determined by using an alignment algorithm such as BlastTM (available at the National Center for Biotechnology Information [NCBI]). A first nucleic acid is "substantially similar" to a second nucleic acid if, when optimally aligned (using the default parameters provided at the NCBI wesite) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about, for example, 80%, 85%, 90% or 95% of the nucleotide bases. Sequence similarity can be determined by comparing the nucleotide sequences of two nucleic acids using the BLASTT sequence analysis software (blastn) available from NCBI. Such comparisons may be made using the software set to default settings (expect filter default, descriptions 500 pairwise, alignments 500, alignment view standard, gap existence cost 11, per residue existence 1, per residue gap cost WO 01/34780 PCT/US00/31254 0.85). Similarly, a first polypeptide is substantially similar to a second polypeptide if they show sequence identity of at least about 75%-90% or greater when optimally aligned and compared using BLAST software (blastp) using default settings.

Oxygenase activity: Enzymes exhibiting oxygenase activity are capable of directly incorporating oxygen into a substrate molecule. Oxygenases can be either dioxygenases, in which case the oxygenase incorporates two oxygen atoms into the substrate; or, monooxygenases, in which only one oxygen atom is incorporated into the primary substrate to form a hydroxyl or epoxide group. Thus, monooxygenases are referred to sometimes as "hydroxylases." Taxoid oxygenases are a subset of oxygenases that specifically utilize taxoids as substrates.

Oxygenases: Oxygenases are enzymes that display oxygenase activity as described supra. However, all oxygenases do not recognize the same substrates.

Therefore, oxygenase enzyme-activity assays may utilize different substrates depending on the specificity of the particular oxygenase enzyme. One of ordinary skill in the art will appreciate that the spectrophotometry-based assay described below is a representative example of a general oxygenase activity assay, and that direct assays can be used to test oxygenase catalysis directed towards different substrates.

II. Characterization of Oxygenases A. Overview of Experimental Procedures Biochemical studies have indicated that at least the first five oxygenation steps of the Taxol pathway are catalyzed by cytochrome P450 hydroxylases (the remaining three oxygenations are also likely catalyzed by cytochrome P450 enzymes), and that these are slow steps of the reaction pathway and, thus, important candidates for cDNA isolation for the purpose of over-expression in relevant producing organisms to increase Taxol yields (Croteau et al., Curr. Topics Plant Physiol. 15:94-104, 1995; and Hezari et al., Planta Med. 63:291-295, 1997).

Protein purification of cytochrome P450 enzymes from Taxus microsomes (Hefner WO 01/34780 PCT/US00/31254 et al., Methods Enzymol. 272:243-250, 1996), as a basis for cDNA cloning, was not performed because the number of P450 species present, and their known similarity in physical properties (Mihaliak et al., Methods Plant Biochem. 9:261-279, 1993), would almost certainly have prevented bringing the individual proteins to homogeneity for amino acid microsequencing.

Therefore, a strategy based on the differential display ofmRNA-reverse transcription-PCR (DD-RT-PCR) was used for isolating transcriptionally active cytochrome P450s in Taxus cells, which previous biochemical studies had shown to undergo substantial up-regulation of the Taxol pathway 16 hours after induction with methyl jasmonate (Hefner et al., Arch. Biochem. Biophys. 360:62-74, 1998).

Differential display experimental schemes allow for the identification of mRNA species that are up-regulated in response to certain stimulus. Generally, one set of samples is not treated with the stimulant, and a second set of samples is treated with the stimulant. Subsequently, the mRNA from both groups is isolated and amplified.

The mRNA of interest is identified by comparing the mRNA from the stimulated and unstimulated samples. The mRNA that is present only in the stimulated sample appears to represent genes that are activated upon stimulation.

In the experiments described below, mRNA from an untreated cell culture was compared to the mRNA from a culture that had been induced with methyl jasmonate for 16 hours. In order to obtain predominantly induced cytochrome P450 sequences, forward primers were designed based on a conserved proline, phenylalanine, glycine (PFG) motif in plant cytochrome P450 genes. The use of primers directed towards the (PFG) motif in conjunction with the DD-RT-PCRbased strategy revealed roughly 100 differentially expressed species, and the sequences of 100 of these were obtained and analyzed. Of these, 39 represented PCR products containing a cytochrome P450-type sequence. Analysis of these sequences revealed that the C-terminus from 21 different and unique cytochrome P450 genes had been isolated. The 21 nucleic acid sequences amplified (amplicons) and identified as regions encoding oxygenases are shown in SEQ ID NOS: 1-21, respectively.

Twelve amplicons were labeled and used as hybridization probes to screen the methyl jasmonate-induced T. cuspidata cell cDNA library. Screening the T.

WO 01/34780 PCT/US00/31254 cuspidata library allowed identification of nine full-length clones. Four additional clones, which were truncated at the 5'-terminus, were obtained in full-length form using a 5'-RACE (Rapid analysis of cDNA ends) method to acquire the missing sequences. Thus, the initial use of the amplicons, described above, has allowed for the identification of thirteen full-length oxygenases (SEQ ID NOS: 43-55, respectively). Subsequently, various molecular techniques were used to identify an additional 10 full-length cDNAs (SEQ ID NOS: 81-86, respectively) and their corresponding amino acid sequences (SEQ ID NOS: 87-92, respectively).

The full-length oxygenase clones identified through the use of the ampliconbased probes can then be cloned into prokaryotic-based and eukaryotic-based expression systems. Once expressed, the functional competence of the resulting oxygenases can be assessed using the spectrophotomctric assay described below.

The clones that are found to be active using the spectrophotometric assay are at a minimum useful for detecting carbon monoxide. Additionally, in the examples provided below, several of the full-length oxygenase-encoding sequences are shown to have in situ oxygenase activity towards taxoids when expressed in Saccharomyces cerevisiae and baculovirus-Spodoptera cells.

Oxygenases produced by cloned full-length oxygenase-encoding sequences also can be tested for the ability to oxygenate taxoid substrates in vivo. This can be done by feeding taxoid intermediates to transgenic cells expressing the cloned oxygenase-encoding sequences.

B. Cloning of Oxygenases As described supra, a DD-RT-PCR scheme was used for the isolation of transcriptionally active cytochrome P450s in Taxus cells, which previously had been shown to undergo substantial up-regulation of the Taxol pathway 16 hours after induction with methyl jasmonate (Hefner et al., Arch. Biochem. Biophys. 360:62-74, 1998). Because an increase in the relevant enzyme activities resulted from induction (indicating de novo protein synthesis), mRNA from an untreated cell culture was compared to mRNA from a culture that had been so induced for 16 hours. In order to obtain predominantly induced cytochrome P450 sequences, forward primers were designed based on a conserved motif in plant cytochrome P450 genes. Related WO 01/34780 PCT/US00/31254 strategies have been used with other plants (Schopfer and Ebel, Mol. Gen. Genet.

258:315-322, 1998). The proline, phenylalanine, glycine (PFG) motif is a wellconserved region of the heme-binding domain (Durst and Nelson, "Diversity and evolution of plant P450 and P450 reductase," in Durst and O'Keefe Drug Metabolism and Drug Interactions, Freund, UK, 1995, pp. 189-206). The corresponding codons of this region contain only two degenerate positions; thus, a set of only eight non-degenerate primers was necessary to encompass all sequence possibilities (Fig. This PFG motif is located 200-250 bp upstream of the stop codon, and the length of the 3'-untranslated region should range between 100 and 300 bp. Thus, the length of the expected PCR fragments would be in the 300-550 bp range. This DD-RT-PCR-based strategy revealed roughly 100 differentially expressed species, and the sequences of 100 of these were obtained and analyzed.

Of these, 39 represented PCR products containing a cytochrome P450-type sequence. Analysis of these sequences revealed that the C-terminus from 21 different and unique cytochrome P450 genes had been isolated. These DNA fragments (12 thus far) are being used as labeled hybridization probes to screen the methyl jasmonate-induced T. cuspidata cell cDNA library. By this means, nine clones have been obtained in full-length form by screening. Four additional clones, which were truncated at the 5'-terminus, were obtained in full-length form using a RACE (Rapid analysis of cDNA ends) method to acquire the missing C. Sequence Analysis The full-length oxygenase sequences initially obtained (using 12 partial sequence probes) were compared pairwise. It was shown that a total of 13 unique sequences (showing less than 85% similarity), designated clones F12, F21, F42, F31, F51, F9, F56, F19, F14, F55, F34, F72, and F10, respectively (SEQ ID NOS: 43-55, respectively) were present Two of the isolated clones, clone F51 (SEQ ID NO: 47) and clone F9 (SEQ ID NO: 48) were not identical to any of the 21 Cterminal fragments originally found by the DD-RT-PCR cloning strategy, bringing the total number of initially identified unique oxygenase genes, and gene fragments, to 23.

WO 01/34780 PCT/US00/31254 The clones obtained also were compared pairwise to all known plant cytochrome P450 oxygenase sequences in the databases (provided at the NCBI website) (Figs. 5A and 5B) provide a dendrogram of these relationships and a table of pairwise similarity and identity comparisons).

This analysis revealed that 11 of the Taxus clones sorted into one cytochrome P450 family. This large group of related clones seems to resemble most closely the CYP90, CYP85, and CYP88 cytochrome P450 families. Some members of these families are known to be involved in terpenoid metabolism gibberellin (diterpene, C20) and brassinosteroid (triterpene C30) biosynthesis], suggesting that the cytochrome P450 clones obtained from Taxus could be involved in the biosynthesis of the diterpenoid Taxol. Table 1 lists accession numbers of relevant sequences and related information. Outlying clones FI0 (SEQ ID NO:55) and F34 (SEQ ID NO: 53) are related more closely to CYP family 82 (phenylpropanoid metabolism) and CYP family 92 (unknown function), respectively.

After the initial 13 full-length clones were identified, six more were isolated.

Thus, the total number of full-length oxygenase clones identified is nineteen. A dendrogram showing the relationship of all of the identified oxygenase clones is provided in Fig. 5C. A table providing both the sequence identity and similarity of the clones is provided in Fig. WO 01/34780 WO 0134780PCTIUSOO/3 1254 Table 1 Closest Relatives to Taxus Cytochrome P450 Sequences Family Description Clones That Are Similar Arabidopsis thaliana GenEMBL X87367 mRNA F9, F12, F14, F19, F21, F31, (1608bp); GenEMBL X87368 gene (4937 bp). F42, F5 1, F55, F56, and P72 Szekeres et al., "Brassinosteroids rescuee (SEQ ID NOS: 48,43, 51, deficiency of CYP90, a cytochrome P450, controlling 44, 46 45, 47, 52, 49, and 54, cell elongation and de-etiolation in Arabidopsis," Cell respectively) 85:171-182 (1996).

Solanwn lycopersicum (tomato) (also Lycopersicon F9, F12, F 14, Fl19, P21, F31, esculentum) GenEMBL U54770 (1395 bp). Bishop et F42, F5 1, F55, F56, and P72 al., "The tomato dwarf gene isolated by heterologous (SEQ ID NOS: 48, 43, 51, transposon tagging encodes the first member of a new 44 46, 45, 47, 52, 49, and 54, family of cytochrome P450," Plant Cell 8:959-969 respectively) (1996).

Zea mays GenEMBL U32579 (1724 bp). Winkler and F9, P12, P14, P9,1F21, 31, CYP88A1 Helentjaris, "The maize dwarf3 gene encodes a P42, F5 1, F55, F56, and P72 cytochrome P450-mediated early step in gibberelli (SEQ ID NOS: 48,43, 51, biosntheis, Plat Cll 71307131 (195).44, 46, 45, 47, 52, 49, and 54, biosnthsis" PantCell7:107-317(195).respectively) Pisum sativwn (pea) GenEMBL U29333 (1763 bp).

CYP82A1 Frank et al., "Cloning of phenyipropanoid pathway Outlying Clone Fl0 P450 monooxygenases expressed in Pisum sattvum," (SEQ ID NO: unpublished.

Glycine max (soybean) GenEMBL Y10491 (1757 bp).

CYP82A2 Schopfer and Ebel, "Identification of elicitor-induced Outlying Clone F34 cytochrome P450s of soybean (Glycine max using (SEQ ID NO: 53) differential display of mRNA," Mol. Gen. Gene.

258:315-322 (1998).

Nicoliana tabacum (tobacco) GenEMBL X95342 CYP92A2 (I 628bp). Czemnic et al., "Characterization of hsr20l Outlying Clone F34 and hsr2 15, two tobacco genes preferentially SQINO53 expressed during the hypersensitive rcaction provoked (SQINO53 by phytopathogenic. bacteria," unpublished.

WO 01/34780 PCT/US00/31254 D. Functional Expression Functional cytochrome P450 expression can be obtained by using the plasmid in yeast (Saccharomyces cerevisiae) engineered to co-express one or the other of a cytochrome P450 reductase from Arabidopsis thaliana; the plantderived reductase is important for efficient electron transfer to the cytochrome (Pompon et al., Methods Enzymol. 272:51-64, 1999).

Since a functional P450 cytochrome, in the appropriately reduced form, will bind competently to carbon monoxide and give a characteristic CO-difference spectrum (Omura and Sato, J. Biol. Chem. 239:2370-2378, 1964), a spectrophotometric means for assessing, and quantitatively estimating, the presence of functional recombinant cytochrome P450 in transformed yeast cells by in situ (in vivo) measurement was developed. CO-sensor of the 19 full-length cytochrome P450 clones from Taxus, thus far obtained, ten have yielded detectable COdifference spectra (Table It is expected that cytochrome P450 clones that do not yield reliable expression in S. cerevisiae can be transferred to, expressed in, and confirmed by CO-difference spectrum utilizing alternative prokaryotic and eukaryotic systems. These alternative expression systems for cytochrome P450 genes include the yeast Pichiapastoris, for which expression vectors and hosts are commercially available (Invitrogen, Carlsbad, CA), as well as established E. coli and baculovirus-insect cell systems for which general expression procedures have been described (Barnes, Methods Enzymol. 272:1-14, 1996; Gonzalez et al., Methods Enzymol. 206:93-99, 1991; Lee et al., Methods Enzymol. 272:86-98, 1996; and Lupien et al., Arch. Biochem. Biophys. 368:181-192, 1999).

Clones that prove to be capable of binding to CO are useful at least for detecting CO in various samples. Further testing of the recombinantly expressed clones may prove that they are additionally useful for adding one or more oxygen atoms to taxoid substrates.

WO 01/34780 PCT/US0O/31254 E. In vivo Assays of Yeast Cells Expressing Recombinant Oxygenases 1. Use of substrates [20-'H3jtaxa4(5),11(12)-diene or 120- 3

H

2 ]taxa-4(20,1 1(12)-dien-5a-ol Transformed yeast cells that functionally express a recombinant cytochrome P450 gene from Taxus (by CO-difference spectrum) can be tested in vivo for their ability to oxygenate (hydroxylate or epoxidize) taxoid substrates fed exogenously to the cells, thereby eliminating the need for microsome isolation for preliminary in vitro assays.

Accordingly, several clones of the available full-length clones were expressed in induced yeast host cells. These cells were fed [20- 3

H

3 ]taxa- 4(5),11(12)-diene or [20-'H 2 ]taxa-4(20,1 (12)-dien-5a-ol in separate incubations and compared to untransformed controls similarly fed (and that were shown to be inactive with taxoid substrates). The extracts resulting from these incubations were analyzed by radio-HPLC, and the clones that yielded a product are shown below in Table 2.

Representative HPLC traces are shown in Figures 6A-6C. Representative GC-MS (gas chromatography-mass spectrometry) analyses of the products from an incubation are shown in Figs. 6D and 6E. The results shown in Figs. 6A-6E confirm that two distinctly different taxadien-diols derived from taxadien-5a-ol were formed, one yielding the expected parent ion at P+ m/z 304, and the other less stable to the conditions of the analysis in losing water readily to yield the highest mass ion at m/z 286 (P+-H 2 0).

2. Use of substrate [20-3H2] taxa-4(20), 11(12)-dien-5a-yl acetate Transformed yeast cells that functionally express a recombinant cytochrome P450 gene from Taxus (by CO-difference spectrum) were tested in vivo for their ability to oxygenate (hydroxylate or epoxidize) taxoid substrates fed exogenously, thereby eliminating the need for microsome isolation for such a preliminary in vitro assay. The clones indicated in Table 2, below, were induced in yeast host cells that WO 01/34780 PCT/US00/31254 were fed [20-'H 2 ]taxa-4(20), 11(12)-dien-5a-yl acetate in separate incubations and compared to untransformed controls similarly fed (and that were shown to be inactive with taxoid substrates). The ether extracts resulting from these incubations were analyzed by radio-HPLC. Several clones converted the acetate substrate to a more polar product.

WO 01/34780 WO 0134780PCT/USOO/31254 Table 2 Full-length Probe name Co assayed with product identified nO:e nt/aa IDaa ISE Nme (SEQa ID (E DN O: dftsecaIL)pa F12 an! 4 Taxadiene No 43/56 11/32 Taxadien5axol Taxadienyl Ac F21 cbl Taxadiene No 44/5 7 10/31 Taxadien5ctol Taxadienyl Ac No F31 ab2 Taxadiene No 46/59 1122 Taxadien5aol No Taxadienyl Ac No F42 ai2 -Taxadiene No 45/5 8 5/26 Taxadien5nxol No F51 Lib. Screen Taxadiene No 47/60 Taxadien5czol Ac 4+ F72 cm2 Taxadiene No 54/67 19/40 Taxadien5otol Taxadienyl Ac F82 dl Taxadiene No 81/87 20/41 'raxadien5aol Ac -4-4 F9 Lib. Screen Taxadiene No 48/61 Taxadien5aol Ac F56 e12 -Taxadiene No 49/62 8/29 Taxadien5aol No F14 eal Taxadiene No 5 1/64 13/34 'Taxadien5aol ______Taxadienyl Ac F19 dsl Taxadiene No 50/63 14/35 Taxadien~aol No cf2 -Taxadiene No 52/65 6/27 Taxadien5czol No F16 ael I+ Taxadiene No 82/88 2/23 Taxadien5aol Taxadienyl Ac F7 cjl I Taxadiene No 83/89 7/28 Taxadien5aol Taxadienyl Ac F23 dii Taxadine No 84/90 15/36 Taxadien5axol No FIO bai Taxadiene No 55/68 17/3 8 Taxadien5aol No WO 01/34780 PCT/US00/31254 F34 dul Taxadiene No 53/66 Taxadien5aol No df 12/33 85/91 F38 ad6 86/92 16/37 Additional testing of the clone F14 (SEQ ID NO: 64) metabolite was conducted. The metabolite isolated by HPLC was subjected to GC-MS analysis and shown to possess a retention time (compared to the starting material) and mass spectrum that were consistent with respective data obtained from a taxadien-diol monoacetate [the parent ion was observed at m/z 346 (taxadienyl acetate (MW 330) plus 0) with diagnostic ions at m/z 328 (P+-H 2 313 (P+-H 2 0-CH 3 286

(P+-CH

3 COOH), 271 (P+-CH3COOH-CH 3 268 (P+-CH 3

COOH-H

2 0) and 253

(P+-CH

3

COOH-CH

3

-H

2 Preparative-scale incubations of the transformed yeast harboring clone F14 (SEQ ID NO: 51), with the taxadien-5a-yl acetate substrate, yielded the HPLCbased isolation of about 100 ig of the unknown diol monoacetate purity by GC) for NMR analysis. Since all of the 'H resonances oftaxadien-4(20),1 1(12)- (and of the acetate ester) had been assigned previously (Hefner et al., Chem. and Biol. 3:479-489, 1996), elucidation of the structure of the unknown diol monoacetate was accomplished by 'H detection experiments (sample-size-limited direct 3 C measurements).

The 'H-NMR spectrum is illustrated in Fig. 7, and Table 3, below, lists the complete 'H assignments along with their respective one-carbon correlated 3

C

assignments as determined indirectly from hereronuclear single quantum coherence (HSQC; Fig. The assignments are consistent with those of other known taxadien monool and diol derivatives. For example, chemical shifts for CS (8 75.9, C5; 6 5.47, H5) and C10 (5 67.2, C10; 8 4.9 H10) are assigned as oxy-methines. The shifts for C20 (6 111.6, C20; 8 5.07, H20, exo; 8 4.67, H20, endo) are consistent with the exocyclic methylene observed in other taxa-4(20), 11(12)-dienes. Other characteristic shifts are observed for H7a (8 1.84), H19 methyl (8 0.56), H3 (8 2.84), and the gem-dimethyls H16 (6 1.14, exo) and H17 (6 1.59, endo).

WO 01/34780 WO 0134780PCTIUSOO/31254 Table 3 Complete 'H-NMR assignments and one-bond correlated 1 3 C assignments (as measured indirectly from HSQC) for the biosynthetic product derived from taxadien-Sct-yl acetate by the cytochrome P450 expressed from clone F14. For position numbering, see Fig. I.

Position Carbon a-proton P-proton number 43.9 28 35.9 75.9 27.9 33.6 47.6 67.2 30.3 22.7 31.8 25.3 20.7 21.4 111.6 1.59 1.53 1.47 2.84 1.66 1.94 1.42 4.9 1.8 1.26 5.47 1.55 0.9 2.21 2.26 1.96 1. 14 (exo) 1.59 (en~do 1.71 5.07 (exo)q 0.66 4.67 (endo) 21 1.66 21 (acetate) The 2D-TOCS Y spectra (Figs. 9A and 10) complemented the H-SQC data and permitted additional regiochemnical assignments. The H5 proton (8 5.47) (Figs.

IOA and IlOE) was correlated strongly with H6 (S 1.66, 8 1.5 5) and H7 (S 1.94, 8 0.9) protons but had no appreciable coupling to either of the H20 signals (8 5.07, 8 26 WO 01/34780 PCT/US00/31254 4.67) or to H3 (8 2.84), which is a common feature observed with taxadiene derivatives. The spin system defined in part by H3 (6 2.84), H2 (8 1.47 and 8 1.53), HI (8 1.59), H13 (8 1.80,8 2.26), and H14 (8 1.26, 8 1.96) was apparent in Figs.

and 10E. The H18 allylic methyl (8 1.71) also displayed a weak correlation with H13. In contrast to the extended spin correlations noted in Fig. 10D, the H9 (8 1.42, 8 2.21) and H10 (8 4.9) signals formed an isolated spin system (see Fig. which included the HIO hydroxyl (8 0.85). A correlation also was observed between the two gem-dimethyl signals (8 1.14 and 8 1.59), which was consistent with the spectra of other taxadiene derivatives.

ROESY (Rotational nuclear Overhauser Effect SpectroscopY) is useful for determining which signals arise from protons which are close in space but not closely connected by chemical bonds. Therefore, 2D-ROESY spectra (Figs. 9B and 11) were used to confirm the regiochemical assignments and to assess relative stereochemistry (Several of these n.O.e correlations are listed in Table 'H-'H TOCSY (TOtal Correlated SpectroscopY) is useful for determining which signals arise from protons within a spin system, especially when the multiplets overlap or there is extensive second order coupling. The 2D-TOCSY (total correlation spectrum) described herein, showed that a second heteroatom was introduced into the C9-C10 fragment, but the regiochemistry was ambiguous based on this single measurement. The 2D-ROESY confirmed that oxidation had occurred at C10 and placed the C10 hydroxyl in the P-orientation. This assignment also was supported by an observed n.O.e between the H10 proton (8 4.90) (Fig. 11B) and the allylic methyl, H 18 (8 1.71), which is consistent with an a-configuration for H Additional stereochemical assignments were made by noting correlations between H9P (8 2.21) and the H17 methyl which must be endo (8 1.59) (Fig. 11E), the H19 methyl (8 0.56) which is P-oriented, and the H2p-proton (8 1.53). The other H9 signal (8 1.42) correlated with H19 and the H7p-proton (8 0.90), as well as H10 (8 4.90) (Figs. 11D and 11B). It also was noted that 3 Jm was large (11.7 Hz) between the H9p- and HlOa1-protons, consistent with a nearly axial arrangement for this pair; a smaller coupling (5.3 Hz) between H9a and H10 was consistent with an equatorial configuration between these two protons.

WO 01/34780 PCT/US00/31254 ROESY spectroscopy also was used to confirm the stereochemistry at Moderately strong correlations were seen between H5 (8 5.47) (see Table 4 and Fig.

11A) and both C6 signals (8 1.66, 5 1.55), consistent with an equatorial orientation for H5. The 3 JHH coupling was quite small 3 Hz) between H5 and all other scalarcoupled partners, providing further evidence for the adopted equatorial orientation of A moderately strong n.O.e between H5 and H20exo was noted, but there were no n.O.e correlations observed between H5 and other protons on the a-face of the molecule. These results confirmed that H5 was P-configured and that the acetate group was ca-oriented as in the substrate. One other significant structural motif in taxadiene derivatives was the near occlusion of the H3 proton on the a-face due to the unusual folding of the molecule, thereby making the H3 proton (8 2.84) a useful probe for this face. Indeed, n.O.e correlations were observed between H3, H13a, and the allylic methyl HI8 (Table 4 below, and Fig. 11C).

Table 4 n.O.e. Correlations Proton n.0.e. correlations H3 alpha 10(w) 13-a 18 (w) beta 20-exo 6-ab (m) H7 beta 19 9-a 6-ab 7-a (s) H7 alpha 7-b 3 10(m) 21(w) H9 alpha 9-b 7-b 19 9-a OH (w) H9 beta 17 9-a 2-b 19 (w) alpha 7-a 18 9-a 19-b OH (w) HI3 beta 14-b 13-a 18 (vw) 16-exo (m) H14 alpha 3 14-b 13-a (m) H14 beta 14-a 16-exo 1 13-b (m) H16 exo 17-endo 3-b 14-b 1 (w) H19 beta 20-endo 20-exo 7-b 9-ab 2-b 6-b endo 20-exo 3 2-a 19 (w) exo 20-endo 5 (m) This full assignment of the structure confirms the identity of the biosynthetic product as taxa-4(5), 11(12)-dien-5a-acetoxy- 10p-ol, and indicates that a cDNA encoding the cytochrome P450 taxane 10p-hydroxylase has been isolated. This 1494-bp cDNA (SEQ ID NO:51) translates a 497 residue deduced protein of WO 01/34780 PCT/US00/31254 molecular weight 56,690 that bears a typical N-terminal membrane anchor (Brown et al., J. Biol. Chem. 264:4442-4449, 1989), with a hydrophobic insertion segment (Nelson et al., J. Biol. Chem. 263:6038-6050, 1988) and a stop-transfer signal (Sakaguchi et al., EMBO J 6:2425-2431, 1987). The protein possesses all of the conserved motifs anticipated for cytochrome P450 oxygenases, including the oxygen-binding domain (Shimada et al., in Bunabiki Oxygenases and Model Systems, Kluwer, Boston, MA, pp. 195-221, 1997) and the highly conserved hemebinding motif (Durst et al., Drug Metab. Drug Interact. 12:189-206, 1995; and von Wachenfeldt et al., in Ortiz de Montellano Cytochrome P450: Structure, to Mechanism, and Biochemistry, Plenum, New York, NY, pp. 183-223, 1995) with PFG element (aa 435-437).

F. In Vitro Assays of Isolated Enzymes for Taxoid Oxygenase Activity The standard enzyme assay for assessing oxygenase activity of the recombinant cytochrome P450 employed the following conditions: 25 mM HEPES buffer, pH 7.5, 400 pM NADPH, 300 utg protein and 30 pM substrate (taxadiene, taxadienol, or taxadienyl acetate) in a total volume of 1 mL. Samples were incubated at 32*C for 12 hours, after which 1 mL of saturated NaCI solution was added to the reaction mixture, followed by extraction of the product with 2 mL of hexane/ethyl acetate The extracts were dried and dissolved in acetonitrile for product analysis by radio-HPLC [column: Alltech Econosil C18 5 pm particle size (250 mm X 4.6 mm): solvent system A: 0.01% H 3

PO

4 2% acetonitrile, 97.99% H 2 0; solvent system B: 0.01% H 3 P0 4 99.99 acetonitrile; gradient: minutes, 100% A; 5-15 minutes, 0-50% B; 15-55 minutes, 50-100% B; 55-65 minutes, 100% B; 65-70 minutes, 0-100% A; 70-75 minutes, 100% A; flow rate 1 mL/minute; for detection, a radio-chromatography detector (Flow-One®-Beta Series A-100, Radiomatic) was used].

Of the three test substrates B, taxadiene was not converted detectably to an oxygenated product by recombinant cytochrome P450 clone F16 (SEQ ID NO: 93). Of the 5a-ol derivatives, taxa-4(20),l 1(12)-dien-5a-ol was converted most efficiently to a diol product as determined by GC-MS analysis (parent ion indicating WO 01/34780 PCT/US00/31254 a MW of 304). Preparative incubations with taxadienol allowed the generation of ~100 pg of the diol product that was purified by a combination of reversed phase HPLC, as described above, and normal phase TLC (silica gel with toluene/acetone in preparation for structural determination by and 3 C-NMR analysis (500 MHz). Comparison of spectra to those of authentic taxa-4(20), 11(12)-dien-Saol (Hefner et al., Chem. Biol. 3:479-489, 1996) indicated that the product of the clone F16 (SEQ ID NO: 93) cytochrome P450 oxygenase reaction is taxa- 4(20),11(12)-dien-5a,9a-diol. These results indicated that clone F16 (SEQ ID NO: 16) encodes a cytochrome P450 taxane 9a-hydroxylase, likely representing the third to regiospecific hydroxylation step of the Taxol biosynthetic pathway.

Additionally, biochemical studies can be done to determine which diol resides on the Taxol pathway the gene encoding the next pathway step suspected to be responsible for C10 hydroxylation), and to determine which activities (and genes) reside further down the pathway (catalyzing formation of triol, tetraol, pentaol, etc.) but that yield a cytochrome P450 oxygenase capable of catalyzing the hydroxylation of taxadien-5a-ol as an adventitious substrate. Other expression systems also can be tested to obtain functional expression of the remaining clones, and all functional clones are being tested with other taxoid substrates.

It is notable that some of the clones that are capable of transforming taxoid intermediates are from the same, closely related family (see placement of clones F9, F12, F14, and F51 (SEQ ID NOS: 61, 56, 64, and 60) in the dendrogram of Fig.

Outlying clone 34, although it yielded a reliable CO-difference spectrum (confirming a functional cytochrome P450 and its utility for detecting CO), does not transform the taxoid substrates to oxygenated products. However, this clone when expressed in a different expression system may prove to be active against other taxoid substrates.

III. Other Oxygenases of the Taxol Pathway The protocol described above yielded 21 related amplicons. Initial use of twelve amplicons as probes for screening the cDNA library allowed for the isolation and characterization of thirteen oxygenase-encoding DNA sequences.

WO 01/34780 PCT/US00/31254 Subsequently, additional full-length enzymes were isolated. Several of these fulllength sequences were expressed recombinantly and tested in situ, and ten were shown to be capable of binding CO, and, therefore, to be useful for detecting CO (Table Additionally, nine clones were shown to be capable ofhydroxylating taxoid substrates in vivo (Table 2).

There are at least five distinct oxygenases in the Taxol biosynthetic pathway (Hezari et al., Planta Med. 63:291-295, 1997), and the close relationship between the nucleic acid sequences of the 21 amplicons indicates that the remaining amplicon sequences represent partial nucleic acid sequences of the other oxygenases in the Taxol biosynthetic pathway. Hence, the above-described protocol enables the identification and recombinant production of oxygenases corresponding to the fulllength versions of the 21 amplicon sequences provided. Therefore, the following discussion relating to Taxol oxygenases refers to the full-length oxygenases shown in the respective sequence listings, as well as the remaining oxygenases of the Taxol biosynthetic pathway that are identifiable through the use of the amplicon sequences. Furthermore, one of skill in the art will appreciate that the remaining oxygenases can be tested easily for enzymatic activity using "functional assays" such as the spectrophotometric assay described below, and direct assays for catalysis with the appropriate taxoid substrates.

IV. Isolating Oxygenases of the Taxol Biosynthetic Pathway A. Cell Culture Initiation, propagation, and induction of Taxus sp. cell cultures have been previously described (Hefner et al., Arch. Biochem. Biophys. 360:62-75, 1998).

Enzymes and reagents were obtained from United States Biochemical Corp.

(Cleveland, OH), Gibco BRL (Grand Island, NY), Promega (Madison, WI) and New England BioLabs, Inc. (Beverly, MS), and were used according to the manufacturers' instructions. Chemicals were purchased from Sigma Chemical Co.

(St. Louis, MO).

WO 01/34780 PCT/US00/31254 B. Vectors and DNA Manipulation Unless otherwise stated, all routine DNA manipulations and cloning were performed by standard methods (Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). PCR amplifications were performed by established procedures (Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, 1990). DNA was sequenced using Amplitaq T M

DNA

polymerase (Roche, Somerville, New Jersey) and fluorescence cycle sequencing on an Applied Biosystems Inc. PrismTM 373 DNA Sequencer (Perkin-Elmer, Norwalk, CT). The Saccharomyces cerevisiae expression vector pYeDP60 was as described previously (Pompon et al., Methods Enzymol. 272:51-64, 1996).

C. E. coil and Yeast Strains The E. coli strains XLI-Blue MRF' (Stratagene, La Jolla, CA) and TOPIOF' (Invitrogen, Carlsbad, CA), were used for routine cloning and for cloning PCR products, respectively. The yeast strains used for expression each expressed one of two different Arabidopsis thaliana cytochrome P450 reductases, and were designated WAT11 and WAT21, respectively (Pompon et al., Methods Enzymol.

272:51-64, 1996).

D. cDNA Library Construction A cDNA library was prepared from mRNA isolated from T. cuspidata suspension cell cultures, which had been induced to maximal Taxol production with methyl jasmonate for 16 hours. Isolation of total RNA from 1.5 g T. cuspidata cells was developed empirically using a buffer containing 4 M guanidine thiocyanate, mM EDTA, 14 mM 2-mercaptoethanol, and 100 mM Tris-HCI, pH 7.5. Cells were homogenized on ice using a polytron (VWR Scientific, Salt Lake City, UT) (4 X second bursts at setting The homogenate was adjusted to 2 Triton X-100 and allowed to stand 15 minutes on ice, after which an equal volume of 3 M sodium acetate, pH 6.0 was added. After mixing, the solution was incubated on ice for an additional 15 minutes, followed by centrifugation at 15,000 g for 30 minutes at 4 0

C.

WO 01/34780 PCT/US00/31254 The supernatant was mixed with 0.8 volume ofisopropanol and left to stand on ice for 5 minutes. After centrifugation at 15,000 g for 30 minutes at 4 0 C, the resulting pellet was redissolved in 8 mL 20 mM Tris-HCI, pH 8.0, containing 1 mM EDTA, then adjusted to pH 7.0 by addition of 2 mL 2 M NaCI in 250 mM MOPS buffer at pH 7.0. Total RNA was recovered by passing this solution over a nucleic-acidisolation column (Qiagen, Valencia, CA) following the manufacturer's instructions.

Poly(A) RNA was purified by using the OligotexTM mRNA kit following the manufacturer's instructions (Qiagen, Valencia, CA). Messenger RNA prepared in this fashion was used to construct a library using a XZAPIIT-cDNA synthesis kit and ZAP-cDNA gigapack III T gold packaging kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. The isolated mRNA also was used to construct a RACE (Rapid Amplification of cDNA Ends) library using a Marathon cDNA amplification kit (Clontech, Palo Alto, CA).

E. Differential Display of mRNA Differential display of mRNA was performed using the Delta Differential Display Kit (Clontech, Palo Alto, CA) by following the manufacturer's instructions except were noted. Total RNA was isolated as described above from two different Taxus cuspidata suspension cell cultures, one that had been induced with methyl jasmonate 16 hours before RNA isolation and the other that had not been treated uninduced). Cytochrome P450-specific forward primers (Fig. instead of random primers, were used in combination with reverse-anchor-(dT)9N-IN-l primers (where N-l A, G, or C) provided in the kit. The anchor designed by Clontech was added to each P450-specific primer to increase the annealing temperature after the fourth low-stringency PCR cycle; this led to a significant reduction of the background signal. Each cytochrome P450-specific primer was used with the three anchored oligo(dT) primers terminated by each nucleotide. PCR reactions were performed with a RoboCycler T M 96 Temperature Cycler (Stratagene, La Jolla, CA), using one cycle at 94 0 C for 5 minutes, 40 0 C for 5 minutes, 68 0 C for minutes, followed by three cycles at 94C for 30 seconds, 40°C for 30 seconds, 68 0 C for 5 minutes, and 32 cycles at 94 0 C for 20 seconds, 60 0 C for 30 seconds, and 68 0 C for 2 minutes. Finally, the reactions were heated at 68 0 C for 7 minutes. The WO 01/34780 PCT/US00/31254 resulting amplicons were separated on a 6% denaturing polyacrylamide gel (HR- 100, Genomyx Corporation, Foster City, CA) using the LR DNA Sequencer Electrophoresis System (Genomyx Corporation).

Differential display bands of interest were cut from the dried gel, eluted with 100 mL of 10 mM Tris-HCI buffer, pH 8.0, containing 1 mM EDTA, by incubation overnight at 4 0 C. A 5-mL aliquot of the extract was used to re-amplify the cDNA fragment by PCR using the same primers as in the original amplification. The reactions initially were heated to 94C for 2 minutes, then subjected to 30 cycles at 94°C for 1 minute, 60°C for 1 minute, and 68 0 C for 2 minutes. Finally, to facilitate cloning of the PCR product, the reactions were heated at 68 0 C for 7 minutes.

Amplicons were analyzed by agarose gel electrophoresis as before. Bands were excised from the gel and the DNA was extracted from the agarose. This gel-purified cDNA was then transferred into the T/A cloning vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA).

The DD-RT-PCR-based screening revealed about 100 clearly differentially expressed bands, all of which were sequenced and analyzed. Of these, 39 represented PCR products containing cytochrome P450-like sequences. The nucleotide and deduced peptide sequences of these 39 amplicons were compiled using the GCG fragment assembly programs and the sequence-alignment program "Pileup" (Genetics Computer Group, Program Manual for the Wisconsin Package, Version 9, Genetics Computer Group, 575 Science Drive, Madison, WI, 1994). This comparison of cloned sequences revealed that C-terminal fragments from 21 different cytochrome P450 genes had been isolated. These cytochrome P450 sequences were used to prepare hybridization probes in order to isolate the corresponding full-length clones by screening the cDNA library.

F. cDNA Library Screening Initially, 12 probes (SEQ ID NOS: 11, 10, 1, 5, 4, 19, 8, 17, 13, 14, 21, and 6, respectively) were labeled randomly using the Ready-To-GoTM kit (Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer's instructions.

Plaque lifts of the T. cuspidata phage library were made on nylon membranes and were screened using a mixture of two radiolabeled probes. Phage DNA was cross- WO 01/34780 PCT/US00/31254 linked to the nylon membranes by autoclaving on fast cycle for 3 minutes at 120 0

C.

After cooling, the membranes were washed for 5 minutes in 2 X SSC (sodium citrate buffer). Prehybridization was performed for I to 2 hours at 65C in 6 X SSC, containing 0.5% SDS, and 5 X Denhardt's reagent. Hybridization was performed in the same buffer for 20 hours at 65°C. The nylon membranes were washed twice for minutes each in 2 X SSC with 0.1% SDS at room temperature, and twice for 1 hour each in 1 X SSC with 0.1% SDS at 65 0 C. After washing, the membranes were exposed for 17 hours onto Kodak (Rochester, NY) XARTM film at -70 0 C. Positive plaques were purified through one additional round of hybridization. Purified XZAPII clones were excised in vivo as pBluescript II phagemids (Stratagene, La Jolla, CA) and transformed into E. coli SOLR cells. The size of each cDNA insert was determined by PCR using T3 and T7 promoter primers. Inserts kb; of a size necessary to encode a typical cytochrome P450 of 50-60 kDa) were sequenced and sorted into groups based on sequence similarity/identity using the GCG fragment assembly programs (Genetics Computer Group, Program Manual for the Wisconsin Package, Version 9, Genetics Computer Group, 575 Science Drive, Madison, WI, 1994). Each unique sequence was used as a query in database searching using either BLAST or FASTA programs (Genetics Computer Group, Program Manual for the Wisconsin Package, Version 9, Genetics Computer Group, 575 Science Drive, Madison, WI, 1994), to define sequences with significant homology to plant cytochrome P450 sequences. These clones also were compared pairwise at both the nucleic acid and amino acid levels using the "Pileup" and "Gap" programs (Genetics Computer Group, Program Manual for the Wisconsin Package, Version 9, Genetics Computer Group, 575 Science Drive, Madison, WI, 1994).

G. Generation of Full-Length Clones by Of the 13 clones initially examined, full-length sequences of nine were obtained by screening of the T. cuspidata h-phage library with the corresponding probes (clones F12, F21, F31, F42, F51, F72, F9, F56, and F10, respectively (SEQ ID NOS: 43, 44, 46, 45, 47, 54, 48, 49, and 55, respectively)). To obtain the sequence portions of the other four truncated clones F14, F19, F34, and F55 (SEQ ID NOS: 51, 50, 53 and 52, respectively), 5'-RACE was performed using the WO 01/34780 PCT/US00/31254 Marathon cDNA amplification kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. The reverse primers used were: for F14, TCGGTGATTGTAACGGAAGAGC-3' (SEQ ID NO: 69); for F19, CTGGCTITTCCAACGGAGCAT-GAG-3' (SEQ ID NO: 70); for F34, ATTGTTTCTCAGCCCGCGCAGTATG-3' (SEQ ID NO: 71); for F55, TTCTATGACGGAAGAGATG-3' (SEQ ID NO: 72). Using the defined sequences thus acquired, and the previously obtained 3-sequence information, primers corresponding to these terminal regions were designed and the full-length versions of each clone were obtained by amplification with Pfu polymerase (Stratagene, La Jolla, CA) using library cDNA as target. These primers also were designed to contain nucleotide sequences encoding restriction sites that were used to facilitate cloning into the yeast expression vector.

H. cDNA Expression of Cytochrome P450 Enzymes in Yeast Appropriate restriction sites were introduced by standard PCR methods (Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA, 1990) immediately upstream of the ATG start codon and downstream of the stop codon of all full-length cytochrome P450 clones. These modified amplicons were gel-purified, digested with the corresponding restriction enzymes, and then ligated into the expression vector pYeDP60. The vector/insert junctions were sequenced to ensure that no errors had been introduced by the PCR construction. Verified clones were transformed into yeast using the lithium acetate method (Ito et al., J. Bacteriol. 153:163-168, 1983). Isolated transformants were grown to stationary phase in SGI medium (Pompon et al., Methods Enzymol.

272:51-64, 1996), and used as inocula for a large-scale expression culture grown in YPL medium (Pompon et al., Methods Enzymol. 272:51-64, 1996). Approximately 24 hours after induction of cytochrome P450 expression with galactose (to 10% final concentration), a portion of the yeast cell culture was harvested by centrifugation.

One-half of the culture was treated with carbon monoxide, and the cytochrome P450 CO-difference spectrum was recorded directly (using untreated cells as a control) by spectrophotometry (Omura and Sato, J. Biol. Chem. 239:2370-2378, 1964).

WO 01/34780 PCT/US00/31254 This direct, in situ method for demonstrating the presence of functional, recombinant cytochrome P450, and for estimating the quantity of the competent enzyme, also can be applied to other expression systems, including E. coli, Pichia pastoris, insect cells (as described below), and Spodopterafugiperda cells. Of the 13 full-length clones obtained so far, eight exhibit a detectable CO-difference spectrum when the recombinant cytochrome P450 gene product is expressed in this yeast system and assayed by this in situ method.

I. cDNA Expression of Cytochrome P450 Enzymes in Insect Cells As mentioned above, insect cell expression systems, such as the baculovirus- Spodoptera system described below, can be used to express the oxygenases described herein.

For example, the functional identification of the Taxus cuspidata cytochrome P450 clone F 16 was accomplished using the baculovirus-Spodoptera expression system. (The use of this system for the heterologous expression of cytochrome P450 genes has been described previously (Asseffa et al., Arch. Biochem. Biophys.

274:481-490, 1989; Gonzalez et al., Methods Enzymol. 206:93-99, 1991; and Kraus et al., Proc. Natl. Acad. Sci. USA 92:2071-2075, 1995)). For the heterologous expression of clone F16 in Spodopterafugiperda Sf9 cells with the Autographa californica baculovirus expression system, the F 16 cytochrome P450 open reading frame (orf) was amplified by PCR using the F16-pYEDP60 construct as a template.

For PCR, two gene-specific primers were designed that contained, for the purpose of subcloning the F16 orf into the FastBac-1 vector (Life Technologies), a BamHI and a NotI restriction site (forward primer 5'-gggatccATGGCCCTTAAGCAATTGGAAGTTTC-3' (SEQ ID NO:93); reverse primer 5'-ggcggccgcTTAAGATCTGGAATAGAGTTTAATGG-3' (SEQ ID NO:94)). The gel-purified PCR product so obtained was subcloned into the pCR- Blunt vector (Invitrogen, Carlsbad, CA). From the derived recombinant pCR-Blunt vector, the subcloned cytochrome P450 orf was excised using the added restriction sites, and the obtained DNA fragment was ligated into the BamHI/NotI-digested pFastBacl vector (Life Technologies, Grand Island, NY). The sequence and the correct insertion of clone F16 into the pFastBac vector were confirmed by WO 01/34780 PCT/US00/31254 sequencing of the insert. The pFastBac/F 16orf construct was then used for the preparation of the recombinant Bacmid DNA by transformation of the Escherichia coli strain DH 0Bac (Life Technologies). Construction of the recombinant Bacmid DNA and the transfection ofSpodopterafrugiperda Sf9 cells were done according to the manufacturer's protocol.

The Spodopterafrugiperda Sf9 cell cultures were propagated either as adherent monolayer cultures in Grace insect cell culture medium (Life Technologies) supplemented with 10% FCS (Life Technologies) or as suspension cultures in Grace medium containing 10% FCS and 0.1% Pluronic F-68 (Sigman, St.

Louis, MO). The adherent cell cultures were maintained in a chamber at 28 0 C. The suspension cultures were incubated in a shaker at 28 0 C at 140 rpm. The adherent cell cultures were grown in T25 tissue culture flasks (Nalgene Nuc, Rochester, NY) with passage of one-third to one-half of the culture every 2 to 3 days. For heterologous protein production, the cultures were grown as suspensions. The cells from two tissue culture flasks (80-90% confluent) were added to 50 mL of standard suspension insect culture medium in a 100 mL conical flask, and were incubated as above until a cell density of-2 X 106 cells/mL was reached. The cells were collected by centrifugation at room temperature at 140 g for 10 minutes. The resulting cell pellet was resuspended in 1/10 of the original volume with fresh medium.

For the functional characterization of clone F 16, the recombinant baculovirus carrying the cytochrome P450 clone F16 ORF was coexpressed with a recombinant baculovirus carrying the Taxus NADPH:cytochrome P450 reductase gene. To the insect cell suspension, the two recombinant baculoviruses were added at a multiplicity of infection of 1-5. The viral titers were determined according to the End-Point Dilution method (O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, New York, NY, Freeman and Company, 1992). For infection, the cells were incubated for 1 hour at 28 0 C and 80 rpm. The cell culture volume was brought to 50 mL with standard cell culture medium, and hemin (Sigma) was added to a final concentration of 2 pg/mL. The infected cells were incubated for 48 hours in a gyratory shaker at 28 0 C and 140 rpm. The infected insect cells were harvested from the cell culture medium by centrifugation as described above, and WO 01/34780 PCT/US0O/31254 washed twice with PBS (50 mM KH 2 PO4, pH 7.5, 0.9% NaCI). The cell pellet so obtained was resuspended in 5 mL of HEPES/DTT Buffer (25 mM HEPES, pH 1 mM DTT). The cells were lysed by mild sonication (VirSonic, Virtis Company, Gardiner, NY), the cell debris was removed by centrifugation at 5,000 g for minutes at 4°C, and the resulting supernatant was collected for use in enzyme assays.

J. Assay of Recombinant Cytochrome P450 Activity Toward Taxoid Substrates Isolated transformants for each full-length cytochrome P450 clone shown to express a functional enzyme by CO-difference spectrum (ten clones) were grown to stationary phase in 2 mL SGI medium at 30 0 C and used to inoculate a expression culture (in YPL medium). Approximately 8 hours after induction, cells were harvested by centrifugation (10 minutes at 1500 rpm), and the pellet was resuspended in 2 mL of fresh YPL medium.

To eliminate additional complication and uncertainty associated with microsome isolations for in vitro assays, 106 dpm of [20- 3

H

3 ]taxa-4(5), 1(12)-diene (16 Ci/mol) or [20-'H 2 ]taxa-4(20), 11(12)-dien-5-a-ol (4.0 Ci/mol), or other taxoid substrate were added directly to the cell suspension to assay conversion in vivo.

After 12 hours of incubation at 30 0 C with agitation (250 rpm), the mixture was treated for 15 minutes in a sonication bath and extracted 3 times with 2 mL diethyl ether to insure isolation of the biosynthetic products. These ether extracts, containing residual substrate and derived product(s), were concentrated to dryness, resuspended in 200 pL ofCH 3 CN, and filtered. These samples were analyzed by radio-HPLC (Hefner et al., Chemistry and Biology 3:479-489, 1996) using a 4.6 mm i.d. X 250 mm column of Econosil Cl 8, 5 p (Alltech, Deerfield, IL) with a gradient of CH 3 CN in H 2 0 from 0% to 85% (10 minutes at 1 mL/minute), then to 100% CH 3 CN over 40 minutes.

The foregoing method is capable of separating taxoids ranging in polarity from taxadiene to approximately that oftaxadien-hexaol. For confirmation of product type, gas chromatography-mass spectrometry (GC-MS) or liquid WO 01/34780 PCT/US0O/31254 chromatography-mass spectrometry (LC-MS) is employed, depending on the volatility of the product.

In the present example, of the eight clones confirmed to be functional by CO difference spectra, four exhibited a hydroxylated product in situ when incubated with K. Substrate Preparation The syntheses of [20-'H 3 ]taxa-4(5), 1 (12)-diene (16 Ci/mol) and 3

H

2 ]taxa-4(20),11(12)-dien-5a-ol (4.0 Ci/mol) have been described elsewhere (Hefner et al., Chemistry and Biology 3:479-489, 1996; and Rubenstein et al., J.

Org. Chem. 60:7215-7223, 1995, respectively). Other taxane substrates (diols, triols, and tetraols of taxadiene) needed to monitor more advanced cytochrome P450-mediated bioconversions are generated by incubating radiolabeled taxa- 4(20), 1l(12)-dien-5a-ol with isolated T. canadensis microsomes, or appropriate recombinant cytochrome P450 enzymes, and separating the products by preparative (radio)HPLC. Taxusin (5a,9a,100,13a-tetraacetoxy-taxa-4(20), 11(12)-diene) is isolated from Taxus heartwood and purified by standard chromatographic procedures (De Case De Marcano et al., Chem. Commun. 1282-1294, 1969).

Following deacetylation and reacetylation with 4 C] acetic anhydride, this labeled substrate is used to monitor enzymatic hydroxylation at C1, C2, and C7 and epoxidation at C4-C20. 2a-Isobutyryloxy-5a, 7a, 101-triaacetoxy-taxa- 4(20),11(12)-diene, isolated from the same source (De Case De Marcano et al., Chem. Commun. 1282-1294, 1969), can be modified similarly to provide a substrate for monitoring hydroxylation at C9 and C13. If taxa-4(20), 1(12)-dien-5a-ol is hydroxylated at C 10 as an early step, then the surrogate substrates for examining enzymatic oxygenation at all relevant positions of the taxane ring can be procured.

L. NMR Spectrometry All NMR spectra were recorded on a Varian Inova-500 NMR spectrometer operating at 18 0 C using a very sensitive 5 mm pulsed-field-gradient 'H indirectdetection probe. The taxadien-diol monoacetate was dissolved in C 6

D

6 to a final concentration of about 300 gM. A 2D-TOCSY spectrum was acquired using a z- WO 01/34780 PCT/US00/31254 filtered DIPSI mixing sequence, a 60 msec mixing time, 10 kHz spin-lock field, 16 repetitions, 256 (t1) x 2048 (rt) complex points, and 6500 Hz sweep in each dimension. The 2D-ROESY spectrum was acquired using a z-filtered mixing sequence with a 409 msec mixing time, 4 kHz spin-lock field, 128 repetitions, 256 (11) x 2048 (t2) complex points, and 6500 Hz sweep in each dimension. A 2D- HSQC spectrum was acquired using 256 repetitions, 128 (t1) x 1024 (t2) complex points, and 6500 Hz in F2 and 15000 Hz in Fl. The time between repetitions was seconds for these experiments. Data were processed using the Varian, Inc.

VNMR software, version 6.1C. The final data size, after linear-prediction in (t1) and zero-filling in both dimensions, was 1024(FI) x 2048(F2) complex points for all experiments.

EXAMPLES

1. Oxygenase Protein and Nucleic acid Sequences As described above, the invention provides oxygenases and oxygenasespecific nucleic acid sequences. With the provision herein of these oxygenase sequences, the polymerase chain reaction (PCR) may be utilized as a preferred method for identifying and producing nucleic acid sequences encoding the oxygenases. For example, PCR amplification of the oxygenase sequences may be accomplished either by direct PCR from a plant cDNA library or by Reverse- Transcription PCR (RT-PCR) using RNA extracted from plant cells as a template.

Oxygenase sequences may be amplified from plant genomic libraries, or plant genomic DNA. Methods and conditions for both direct PCR and RT-PCR are known in the art and are described in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990.

The selection of PCR primers is made according to the portions of the cDNA (or gene) that are to be amplified. Primers may be chosen to amplify small segments of the cDNA, the open reading frame, the entire cDNA molecule or the entire gene sequence. Variations in amplification conditions may be required to accommodate primers of differing lengths; such considerations are well known in the art and are discussed in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990; Sambrook et al. (eds), Molecular Cloning: A WO 01/34780 PCT/US00/31254 Laboratory Manual 2nd ed., vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York (with periodic updates), 1987. By way of example, the cDNA molecules corresponding to additional oxygenases may be amplified using primers directed toward regions of homology between the 5' and 3' ends of the full-length clone such as the one shown in SEQ ID NO: 43 sequences. Example primers for such a reaction are: primer 1: 5'-CCI CCI GGI AAI ITI- 3' (SEQ ID NO. 81) primer 2: 5'-ICC I(G/C)C ICC (G/A)AA IGG-3' (SEQ ID NO. 82) These primers are illustrative only; it will be appreciated by one skilled in the art that many different primers may be derived from the provided nucleic acid sequences. Re-sequencing ofPCR products obtained by these amplification procedures is recommended to facilitate confirmation of the amplified sequence and to provide information on natural variation between oxygenase sequences.

Oligonucleotides derived from the oxygenase sequence may be used in such sequencing methods.

Oligonucleotides that are derived from the oxygenase sequences are encompassed within the scope of the present invention. Preferably, such oligonucleotide primers comprise a sequence of at least 10-20 consecutive nucleotides of the oxygenase sequences. To enhance amplification specificity, oligonucleotide primers comprising at least 15, 20, 25, 30, 35, 40, 45 or consecutive nucleotides of these sequences also may be used.

A. Oxygenases in Other Plant Species Orthologs of the oxygenase genes are present in a number of other members of the Taxus genus. With the provision herein of the oxygenase nucleic acid sequences, the cloning by standard methods of cDNAs and genes that encode oxygenase orthologs in these other species is now enabled. As described above, orthologs of the disclosed oxygenase genes have oxygenase biological activity and are typically characterized by possession of at least 50% sequence identity counted over the full-length alignment with the amino acid sequence of the disclosed oxygenase sequences using the NCBI Blast 2.0 (gapped blastp set to default WO 01/34780 PCT/US00/31254 parameters). Proteins with even greater sequence identity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% sequence identity.

Both conventional hybridization and PCR amplification procedures may be utilized to clone sequences encoding oxygenase orthologs. Common to both of these techniques is the hybridization of probes or primers that are derived from the oxygenase nucleic acid sequences. Furthermore, the hybridization may occur in the context of Northern blots, Southern blots, or PCR.

Direct PCR amplification may be performed on cDNA or genomic libraries prepared from the plant species in question, or RT-PCR may be performed using mRNA extracted from the plant cells using standard methods. PCR primers will comprise at least 10 consecutive nucleotides of the oxygenase sequences. One of skill in the art will appreciate that sequence differences between the oxygenase nucleic acid sequence and the target nucleic acid to be amplified may result in lower amplification efficiencies. To compensate for this, longer PCR primers or lower annealing temperatures may be used during the amplification cycle. Whenever lower annealing temperatures are used, sequential rounds of amplification using nested primer pairs may be necessary to enhance specificity.

For conventional hybridization techniques the hybridization probe is preferably conjugated with a detectable label such as a radioactive label, and the probe is preferably at least 10 nucleotides in length. As is well known in the art, increasing the length of hybridization probes tends to give enhanced specificity. The labeled probe derived from the oxygenase nucleic acid sequence may be hybridized to a plant cDNA or genomic library and the hybridization signal detected using methods known in the art. The hybridizing colony or plaque (depending on the type of library used) is purified and the cloned sequence contained in that colony or plaque isolated and characterized.

Orthologs of the oxygenases alternatively may be obtained by immunoscreening of an expression library. With the provision herein of the disclosed oxygenase nucleic acid sequences, the enzymes may be expressed and purified in a heterologous expression system E. coli) and used to raise WO 01/34780 PCT/US00/31254 antibodies (monoclonal or polyclonal) specific for oxygenases. Antibodies also may be raised against synthetic peptides derived from the oxygenase amino acid sequence presented herein. Methods of raising antibodies are well known in the art and are described generally in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Springs Harbor, 1988. Such antibodies can be used to screen an expression cDNA library produced from a plant. This screening will identify the oxygenase ortholog. The selected cDNAs can be confirmed by sequencing and enzyme activity assays.

B. Taxol Oxygenase Variants With the provision of the oxygenase amino acid sequences (SEQ ID NOS: 56-68) and the corresponding cDNA (SEQ ID NOS: 43-55 and 81-86), variants of these sequences now can be created.

Variant oxygenases include proteins that differ in amino acid sequence from the oxygenase sequences disclosed, but that retain oxygenase biological activity.

Such proteins may be produced by manipulating the nucleotide sequence encoding the oxygenase using standard procedures such as site-directed mutagenesis or the polymerase chain reaction. The simplest modifications involve the substitution of one or more amino acids for amino acids having similar biochemical properties.

These so-called "conservative substitutions" are likely to have minimal impact on the activity of the resultant protein. Table 4 shows amino acids that may be substituted for an original amino acid in a protein and that are regarded as conservative substitutions.

WO 01/34780 PCT/US00/31254 Table 4 Original Conservative Residue Substitutions ala Ser arg Lys asn Gin; his asp Glu cys Ser gin Asn glu Asp gly Pro his Asn; gin ile Leu; val leu ile; val lys Arg; gin; glu met Leu; ile phe Met; leu; tyr ser Thr thr Ser trp Tyr tyr Trp; phe val ile; leu More substantial changes in enzymatic function or other features may be obtained by selecting substitutions that are less conservative than those in Table 4, by selecting residues that differ more significantly in their effect on maintaining: the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions that in general are expected to produce the greatest changes in protein properties will be those in which: a hydrophilic residue, seryl or threonyl, is substituted for (or by) a hydrophobic residue, leucyl, isoleucyl, phenylalanyl, valyl or alanyl; a cysteine or proline is substituted for (or by) any other residue; a residue having an electropositive side chain, lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, glutamyl or aspartyl; or a residue having a bulky side chain, phenylalanine, is substituted for (or by) one not having a side chain, glycine. The effects of these amino acid substitutions or deletions or additions may be assessed for oxygenase derivatives by analyzing the ability of the derivative WO 01/34780 PCT/USOO/31254 proteins to catalyse the conversion of one Taxol precursor to another Taxol precursor.

Variant oxygenase cDNA or genes may be produced by standard DNAmutagenesis techniques, for example, M 13 primer mutagenesis. Details of these techniques are provided in Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Ch. 15. By the use of such techniques, variants may be created that differ in minor ways from the oxygenase cDNA or gene sequences, yet that still encode a protein having oxygenase biological activity. DNA molecules and nucleotide sequences that are derivatives of those specifically disclosed herein and that differ from those disclosed by the deletion, addition, or substitution of nucleotides while still encoding a protein having oxygenase biological activity are comprehended by this invention. In their simplest form, such variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced.

Alternatively, the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence in such a way that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence identical or substantially similar to the disclosed oxygenase amino acid sequences. For example, the nineteenth amino acid residue of the oxygenase (Clone F12, SEQ ID NO:43) is alanine. This is encoded in the open reading frame (ORF) by the nucleotide codon triplet GCT. Because of the degeneracy of the genetic code, three other nucleotide codon triplets GCA, GCC, and GCG also code for alanine. Thus, the nucleotide sequence of the ORF can be changed at this position to any of these three codons without affecting the amino acid composition of the encoded protein or the characteristics of the protein. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the cDNA and gene sequences disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences.

Thus, this invention also encompasses nucleic acid sequences that encode the oxygenase protein but that vary from the disclosed nucleic acid sequences by virtue of the degeneracy of the genetic code.

WO 01/34780 PCT/US00/31254 Variants of the oxygenase also may be defined in terms of their sequence identity with the oxygenase amino acid (SEQ ID NOS: 56-68 and 87-92) and nucleic acid sequences (SEQ ID NOS: 43-55 and 81-86). As described above, oxygenases have oxygenase biological activity and share at least 60% sequence identity with the disclosed oxygenase sequences. Nucleic acid sequences that encode such proteins may be readily determined simply by applying the genetic code to the amino acid sequence of the oxygenase, and such nucleic acid molecules may readily be produced by assembling oligonucleotides corresponding to portions of the sequence.

As previously mentioned, another method of identifying variants of the oxygenases is nucleic acid hybridization. Nucleic acid molecules derived from the oxygenase cDNA and gene sequences include molecules that hybridize under various conditions to the disclosed Taxol oxygenase nucleic acid molecules, or fragments thereof.

Nucleic acid duplex or hybrid stability is expressed as the melting temperature at which a probe dissociates from a target DNA. This melting temperature is used to define the required stringency conditions. If sequences are to be identified that are related and substantially identical to the probe, rather than identical, then it is useful to first establish the lowest temperature at which only homologous hybridization occurs with a particular concentration of salt SSC or SSPE). Then, assuming that 1% mismatching results in a 1°C decrease in the Tm, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having 95% identity with the probe are sought, the final wash temperature is decreased by In practice, the change in Tm can be between 0.5 0 C and 1.5°C per 1% mismatch.

Generally, hybridization conditions are classified into categories, for example very high stringency, high stringency, and low stringency. The conditions for probes that are about 600 base pairs or more in length are provided below in three corresponding categories.

WO 01/34780 PCT/US00/31254 Very High Stringency (sequences greater than 90% sequence identity) Hybridization in 5x SSC at 65°C 16 hours Wash twice in 2x SSC at room temp. 15 minutes each Wash twice in 2x SSC at 55°C 20 minutes each High Stringency (detects sequences that share approximately 80% sequence identity) Hybridization in 5x SSC at 420C 16 hours Wash twice in 2x SSC at room temp. 20 minutes each Wash once in 2x SSC at 42°C 30 minutes each Low Stringency (detects sequences that share 70% sequence identity or greater) Hybridization in 6x SSC at room temp. 16 hours Wash twice in 2x SSC at room temp. 20 minutes each The sequences encoding the oxygenases identified through hybridization may be incorporated into transformation vectors and introduced into host cells to produce the respective oxygenase.

2. Introduction of Oxygenases into Plants After a cDNA (or gene) encoding a protein involved in the determination of a particular plant characteristic has been isolated, standard techniques may be used to express the cDNA in transgenic plants in order to modify the particular plant characteristic. The basic approach is to clone the cDNA into a transformation vector, such that the cDNA is operably linked to control sequences a promoter) directing expression of the cDNA in plant cells. The transformation vector is introduced into plant cells by any of various techniques electroporation), and progeny plants containing the introduced cDNA are selected. Preferably all or part of the transformation vector stably integrates into the genome of the plant cell. That part of the transformation vector that integrates into the plant cell and that contains the introduced cDNA and associated sequences for controlling expression (the introduced "transgene") may be referred to as the recombinant expression cassette.

WO 01/34780 PCT/US00/31254 Selection of progeny plants containing the introduced transgene may be made based upon the detection of an altered phenotype. Such a phenotype may result directly from the cDNA cloned into the transformation vector or may be manifest as enhanced resistance to a chemical agent (such as an antibiotic) as a result of the inclusion of a dominant selectable marker gene incorporated into the transformation vector.

Successful examples of the modification of plant characteristics by transformation with cloned cDNA sequences are replete in the technical and scientific literature. Selected examples, which serve to illustrate the knowledge in this field of technology include: U.S. Patent No. 5,571,706 ("Plant Virus Resistance Gene and Methods") U.S. Patent No. 5,677,175 ("Plant Pathogen Induced Proteins") U.S. Patent No. 5,510,471 ("Chimeric Gene for the Transformation of Plants") U.S. Patent No. 5,750,386 ("Pathogen-Resistant Transgenic Plants") U.S. Patent No. 5,597,945 ("Plants Genetically Enhanced for Disease Resistance") U.S. Patent No. 5,589,615 ("Process for the Production of Transgenic Plants with Increased Nutritional Value Via the Expression of Modified 2S Storage Albumins") U.S. Patent No. 5,750,871 ("Transformation and Foreign Gene Expression in Brassica Species") U.S. Patent No. 5,268,526 ("Overexpression of Phytochrome in Transgenic Plants") U.S. Patent No. 5,262,316 ("Genetically Transformed Pepper Plants and Methods for their Production") U.S. Patent No. 5,569,831 ("Transgenic Tomato Plants with Altered Polygalacturonase Isoforms") These examples include descriptions of transformation vector selection, transformation techniques, and the construction of constructs designed to over- WO 01/34780 PCT/US00/31254 express the introduced cDNA. In light of the foregoing and the provision herein of the oxygenase amino acid sequences and nucleic acid sequences, it is thus apparent that one of skill in the art will be able to introduce the cDNAs, or homologous or derivative forms of these molecules, into plants in order to produce plants having enhanced oxygenase activity. Furthermore, the expression of one or more oxygenases in plants may give rise to plants having increased production of Taxol and related compounds.

A. Vector Construction, Choice of Promoters A number of recombinant vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described, including those described in Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant and Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, planttransformation vectors include one or more cloned plant genes (or cDNAs) under the transcriptional control of and 3'-regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter regulatory region a regulatory region controlling inducible or constitutive, environmentally or developmentally regulated, or cell- or tissue-specific expression), a transcription-initiation start site, a ribosome-binding site, an RNA processing signal, a transcription-termination site, and/or a polyadenylation signal.

Examples of constitutive plant promoters that may be useful for expressing the cDNA include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, Odel et al., Nature 313:810, 1985; Dekeyser et al., Plant Cell 2:591, 1990; Terada and Shimamoto, Mol. Gen. Genet. 220:389, 1990; and Benfcy and Chua, Science 250:959-966, 1990); the nopaline synthase promoter (An et al., Plant Physiol.

88:547, 1988); and the octopine synthase promoter (Fromm et al., Plant Cell 1:977, 1989). Agrobacterium-mediated transformation of Taxus species has been accomplished, and the resulting callus cultures have been shown to produce Taxol (Han et al., Plant Science 95: 187-196, 1994). Therefore, it is likely that incorporation of one or more of the described oxygenases under the influence of a WO 01/34780 PCT/US00/31254 strong promoter (like CaMV promoter) would increase production yields of Taxol and related taxoids in such transformed cells.

A variety of plant gene promoters that are regulated in response to environmental, hormonal, chemical, and/or developmental signals also can be used for expression of the cDNA in plant cells, including promoters regulated by: heat (Callis et al., Plant Physiol. 88:965, 1988; Ainley, et al., Plant Mol. Biol. 22:13-23, 1993; and Gilmartin et al., The Plant Cell 4:839-949, 1992); light the pea rbcS-3A promoter, Kuhlemeier et al., Plant Cell 1:471, 1989, and the maize rbcS promoter, Schaffner and Sheen, Plant Cell 3:997, 1991); hormones, such as abscisic acid (Marcotte et al., Plant Cell 1:969, 1989); wounding wunI, Siebertz et al., Plant Cell 1:961, 1989); and chemicals such as methyl jasmonate or salicylic acid (see also Gatz et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:9- 108, 1997).

Alternatively, tissue-specific (root, leaf, flower, and seed, for example) promoters (Carpenter et al., The Plant Cell 4:557-571, 1992; Denis et al., Plant Physiol. 101:1295-1304, 1993; Opperman et al., Science 263:221-223, 1993; Stockhause et al., The Plant Cell 9:479-489, 1997; Roshal et al., Embo. J. 6:1155, 1987; Scherthaner et al., Embo J. 7:1249, 1988; and Bustos et al., Plant Cell 1:839, 1989) can be fused to the coding sequence to obtain a particular expression in respective organs.

Alternatively, the native oxygenase gene promoters may be utilized. With the provision herein of the oxygenase nucleic acid sequences, one of skill in the art will appreciate that standard molecular biology techniques can be used to determine the corresponding promoter sequences. One of skill in the art also will appreciate that less than the entire promoter sequence may be used in order to obtain effective promoter activity. The determination of whether a particular region of this sequence confers effective promoter activity may be ascertained readily by operably linking the selected sequence region to an oxygenase cDNA (in conjunction with suitable 3' regulatory region, such as the NOS 3' regulatory region as discussed below) and determining whether the oxygenase is expressed.

Plant-transformation vectors also may include RNA processing signals, for example, introns, that may be positioned upstream or downstream of the ORF WO 01/34780 PCT/US00/31254 sequence in the transgene. In addition, the expression vectors also may include additional regulatory sequences from the 3'-untranslated region of plant genes, e.g., a 3'-terminator region, to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase (NOS) 3'-terminator regions. The native oxygenase gene 3'-regulatory sequence also may be employed.

Finally, as noted above, plant-transformation vectors also may include dominant selectable marker genes to allow for the ready selection of transformants.

Such genes include those encoding antibiotic-resistance genes resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin, or spectinomycin) and herbicide-resistance genes phosphinothricin acetyloxygenase).

B. Arrangement of Taxol oxygenase Sequence in a Vector The particular arrangement of the oxygenase sequence in the transformation vector is selected according to the type of expression of the sequence that is desired.

In most instances, enhanced oxygenase activity is desired, and the oxygenase ORF is operably linked to a constitutive high-level promoter such as the CaMV 35S promoter. As noted above, enhanced oxygenase activity also may be achieved by introducing into a plant a transformation vector containing a variant form of the oxygenase cDNA or gene, for example a form that varies from the exact nucleotide sequence of the oxygenase ORF, but that encodes a protein retaining an oxygenase biological activity.

C. Transformation and Regeneration Techniques Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells are now routine, and the appropriate transformation technique can be determined by the practitioner. The choice of method varies with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposomemediated transformation; polyethylene glycol (PEG)-mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile WO 01/34780 PCT/US00/31254 bombardment of plant cells; vacuum infiltration; and Agrobacterium tumefaciens (AT)-mediated transformation. Typical procedures for transforming and regenerating plants are described in the patent documents listed at the beginning of this section.

D. Selection of Transformed Plants Following transformation and regeneration of plants with the transformation vector, transformed plants can be selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker confers antibiotic resistance on the seedlings of transformed plants, and selection of transformants can be accomplished by exposing the seedlings to appropriate concentrations of the antibiotic.

After transformed plants are selected and grown to maturity, they can be assayed using the methods described herein to assess production levels of Taxol and related compounds.

3. Production of Recombinant Taxol oxygenase in Heterologous Expression Systems Various yeast strains and yeast-derived vectors are used commonly for the expression ofheterologous proteins. For instance, Pichia pastoris expression systems, obtained from Invitrogen (Carlsbad, California), may be used to practice the present invention. Such systems include suitable Pichia pastoris strains, vectors, reagents, transformants, sequencing primers, and media. Available strains include KM71H (a prototrophic strain), SMD1168H (a prototrophic strain), and SMD1168 (a pep4 mutant strain) (Invitrogen Product Catalogue, 1998, Invitrogen, Carlsbad

CA).

Non-yeast eukaryotic vectors may be used with equal facility for expression of proteins encoded by modified nucleotides according to the invention.

Mammalian vector/host cell systems containing genetic and cellular control elements capable of carrying out transcription, translation, and post-translational modification are well known in the art. Examples of such systems are the wellknown baculovirus system, the ecdysone-inducible expression system that uses WO 01/34780 PCT/US00/31254 regulatory elements from Drosophila melanogaster to allow control of gene expression, and the sindbis viral-expression system that allows high-level expression in a variety of mammalian cell lines, all of which are available from Invitrogen, Carlsbad, California.

The cloned expression vector encoding one or more oxygenases may be transformed into any of various cell types for expression of the cloned nucleotide.

Many different types of cells may be used to express modified nucleic acid molecules. Examples include cells of yeasts, fungi, insects, mammals, and plants, including transformed and non-transformed cells. For instance, common mammalian cells that could be used include HeLa cells, SW-527 cells (ATCC deposit #7940), WISH cells (ATCC deposit #CCL-25), Daudi cells (ATCC deposit #CCL-213), Mandin-Darby bovine kidney cells (ATCC deposit #CCL-22) and Chinese hamster ovary (CHO) cells (ATCC deposit #CRL-2092). Common yeast cells include Pichia pastoris (ATCC deposit #201178) and Saccharomyces cerevisiae (ATCC deposit #46024). Insect cells include cells from Drosophila melanogaster (ATCC deposit #CRL-10191), the cotton bollworm (ATCC deposit #CRL-9281), and Trichoplusia ni egg cell homoflagellates. Fish cells that may be used include those from rainbow trout (ATCC deposit #CLL-55), salmon (ATCC deposit #CRL-1681), and zebrafish (ATCC deposit #CRL-2147). Amphibian cells that may be used include those of the bullfrog, Rana catesbelana (ATCC deposit #CLL-41). Reptile cells that may be used include those from Russell's viper (ATCC deposit #CCL-140). Plant cells that could be used include Chlamydomonas cells (ATCC deposit #30485), Arabidopsis cells (ATCC deposit #54069) and tomato plant cells (ATCC deposit #54003). Many of these cell types are commonly used and are available from the ATCC as well as from commercial suppliers such as Pharmacia (Uppsala, Sweden), and Invitrogen.

Expressed protein may be accumulated within a cell or may be secreted from the cell. Such expressed protein may then be collected and purified. This protein may be characterized for activity and stability and may be used to practice any of the various methods according to the invention.

WO 01/34780 PCT/US00/31254 4. Creation of Oxygenase Specific Binding Agents Antibodies to the oxygenase enzymes, and fragments thereof, of the present invention may be useful for purification of the enzymes. The provision of the oxygenase sequences allows for the production of specific antibody-based binding agents to these enzymes.

Monoclonal or polyclonal antibodies may be produced to an oxygenase, portions of the oxygenase, or variants thereof. Optimally, antibodies raised against epitopes on these antigens will detect the enzyme specifically. That is, antibodies raised against an oxygenase would recognize and bind the oxygenase, and would not substantially recognize or bind to other proteins. The determination that an antibody specifically binds to an antigen is made by any one of a number of standard immunoassay methods; for instance, Western blotting, Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, 2nd ed., vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

To determine that a given antibody preparation (such as a preparation produced in a mouse against SEQ ID NO: 56) specifically detects the oxygenase by Western blotting, total cellular protein is extracted from cells and electrophorcsed on an SDS-polyacrylamide gel. The proteins are transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane. After washing the membrane to remove nonspecifically bound antibodies, the presence of specifically bound antibodies is detected by the use of an anti-mouse antibody conjugated to an enzyme such as alkaline phosphatase; application of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results in the production of a densely blue-colored compound by immuno-localized alkaline phosphatase.

Antibodies that specifically detect an oxygenase will be shown, by this technique, to bind substantially only the oxygenase band (having a position on the gel determined by the molecular weight of the oxygenase). Non-specific binding of the antibody to other proteins may occur and may be detectable as a weaker signal on the Western blot (which can be quantified by automated radiography). The nonspecific nature of this binding will be recognized by one skilled in the art by the WO 01/34780 PCT/US00/31254 weak signal obtained on the Western blot relative to the strong primary signal arising from the specific anti-oxygenase binding.

Antibodies that specifically bind to an oxygenase according to the invention belong to a class of molecules that are referred to herein as "specific binding agents." Specific binding agents capable of specifically binding to the oxygenase of the present invention may include polyclonal antibodies, monoclonal antibodies and fragments of monoclonal antibodies such as Fab, F(ab') 2 and Fv fragments, as well as any other agent capable of specifically binding to one or more epitopes on the proteins.

Substantially pure oxygenase suitable for use as an immunogen can be isolated from transfected cells, transformed cells, or from wild-type cells.

Concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms per milliliter. Alternatively, peptide fragments of an oxygenase may be utilized as immunogens. Such fragments may be synthesized chemically using standard methods, or may be obtained by cleavage of the whole oxygenase enzyme followed by purification of the desired peptide fragments. Peptides as short as three or four amino acids in length are immunogenic when presented to an immune system in the context of a Major Histocompatibility Complex (MHC) molecule, such as MHC class I or MHC class II. Accordingly, peptides comprising at least 3 and preferably at least 4, 5, 6 or more consecutive amino acids of the disclosed oxygenase amino acid sequences may be employed as immunogens for producing antibodies.

Because naturally occurring epitopes on proteins frequently comprise amino acid residues that are not adjacently arranged in the peptide when the peptide sequence is viewed as a linear molecule, it may be advantageous to utilize longer peptide fragments from the oxygenase amino acid sequences for producing antibodies. Thus, for example, peptides that comprise at least 10, 15, 20, 25, or consecutive amino acid residues of the amino acid sequence may be employed.

Monoclonal or polyclonal antibodies to the intact oxygenase, or peptide fragments thereof may be prepared as described below.

WO 01/34780 PCT/US00/31254 A. Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to any of various epitopes of the oxygenase enzymes that are identified and isolated as described herein can be prepared from murine hybridomas according to the classic method of Kohler Milstein, Nature 256:495, 1975, or a derivative method thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA (enzyme-linked immunosorbent assay, as originally described by Engvall, Enzymol. 70:419, 1980, or a derivative method thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988.

B. Polyclonal Antibody Production by Immunization Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified, to enhance immunogenicity.

Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than other molecules and may require the use of carriers and an adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low-titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appear to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al., J. Clin. Endocrinol. Metab. 33:988-991, 1971.

WO 01/34780 PCT/USOO/31254 Booster injections can be given at regular intervals, and antiserum harvested when the antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al., in Wier Handbook of Experimental Immunology, Chapter 19, Blackwell, 1973. A plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/mL of serum (about 12 pM).

Affinity of the antisera for the antigen is determined by preparing competitive binding curves using conventional methods.

C. Antibodies Raised by Injection of cDNA Antibodies may be raised against an oxygenase of the present invention by subcutaneous injection of a DNA vector that expresses the enzymes in laboratory animals, such as mice. Delivery of the recombinant vector into the animals may be achieved using a hand-held form of the "Biolistic" system (Sanford et al., Particulate Sci. Technol. 5:27-37, 1987, as described by Tang et al., Nature (London) 356:153-154, 1992). Expression vectors suitable for this purpose may include those that express the cDNA of the enzyme under the transcriptional control of either the human p-actin promoter or the cytomegalovirus (CMV) promoter.

Methods of administering naked DNA to animals in a manner resulting in expression of the DNA in the body of the animal are well known and are described, for example, in U.S. Patent Nos. 5,620,896 ("DNA Vaccines Against Rotavirus Infections"); 5,643,578 ("Immunization by Inoculation of DNA Transcription Unit"); and 5,593,972 ("Genetic Immunization"), and references cited therein.

D. Antibody Fragments Antibody fragments may be used in place of whole antibodies and may be readily expressed in prokaryotic host cells. Methods of making and using immunologically effective portions of monoclonal antibodies, also referred to as "antibody fragments," are well known and include those described in Better Horowitz, Methods Enzymol. 178:476-496, 1989; Glockshuber et al. Biochemistry 29:1362-1367, 1990; and U.S. Patent Nos. 5,648,237 ("Expression of Functional Antibody Fragments"); 4,946,778 ("Single Polypeptide Chain Binding Molecules"); WO 01/34780 PCT/US00/31254 and 5,455,030 ("Immunotherapy Using Single Chain Polypeptide Binding Molecules"), and references cited therein.

Taxol Production in vivo The creation of recombinant vectors and transgenic organisms expressing the vectors are important for controlling the production ofoxygenases. These vectors can be used to decrease oxygenase production, or to increase oxygenase production.

A decrease in oxygenase production likely will result from the inclusion of an antisense sequence or a catalytic nucleic acid sequence that targets the oxygenase encoding nucleic acid sequence. Conversely, increased production of oxygenase can be achieved by including at least one additional oxygenase encoding sequence in the vector. These vectors can be introduced into a host cell, thereby altering oxygenase production. In the case of increased production, the resulting oxygenase may be used in in vitro systems, as well as in vivo for increased production of Taxol, other taxoids, intermediates of the Taxol biosynthetic pathway, and other products.

Increased production of Taxol and related taxoids in vivo can be accomplished by transforming a host cell, such as one derived from the Taxus genus, with a vector containing one or more nucleic acid sequences encoding one or more oxygenases. Furthermore, the heterologous or homologous oxygenase sequences can be placed under the control of a constitutive promoter, or an inducible promoter.

This will lead to the increased production of oxygenase, thus eliminating any ratelimiting effect on Taxol production caused by the expression and/or activity level of the oxygenase.

6. Taxol Production in vitro Currently, Taxol is produced by a semisynthetic method described in Hezari and Croteau, Planta Medica 63:291-295, 1997. This method involves extracting deacetyl-baccatin III, or baccatin III, intermediates in the Taxol biosynthetic pathway, and then finishing the production of Taxol using in vitro techniques. As more enzymes are identified in the Taxol biosynthetic pathway, it may become possible to completely synthesize Taxol in vitro, or at least increase the number of steps that can be performed in vitro. Hence, the oxygenases of the present invention WO 01/34780 PCT/US00/31254 may be used to facilitate the production of Taxol and related taxoids in synthetic or semi-synthetic methods. Accordingly, the present invention enables the production of transgenic organisms that not only produce increased levels of Taxol, but also transgenic organisms that produce increased levels of important intermediates, such as 10-deacctyl-baccatin III and baccatin III.

7. Alternative Substrates for Use in Assessing Taxoid Oxygenases Activity The order of oxygenation reactions on the taxane (taxadiene) nucleus en route to Taxol is not precisely known. However, based on comparison of the structures of the several hundred naturally-occurring taxanes (Kingston et al., The Taxane Diterpenoids, in Herz et al. Progress in the Chemistry of Organic Natural Products, Springer-Verlag, New York, Vol. 61, p. 206, 1993; and Baloglu et al., J Nat. Prod 62:1448-1472, 1999), it can be deduced from relative abundances of taxoids with oxygen substitution at each position (Floss et al., Biosynthesis ofTaxol, in Suffness Taxol: Science and Applications, CRC Press, Boca Raton, FL, pp. 191-208, 1995) that oxygens at C5 (carbon numbers shown in Fig.) and C 10 are introduced first, followed by oxygenation at C2 and C9 (could be either order), than at C13. Oxygenations at C7 and Cl of the taxane nucleus are considered to be very late introductions, possibly occurring after oxetane ring formation; however, epoxidation (at C4/C20) and oxetane formation seemingly must precede oxidation of the C9 hydroxyl to a carbonyl (Floss et al., Biosynthesis of Taxol, in Suffness Taxol: Science and Applications, CRC Press, Boca Raton, FL, pp. 191-208, 1995). Evidence from cell-free enzyme studies with Taxus microsomes (Hezari et al., Planta Medica 63:291-295, 1997) and in vivo feeding studies with Taxus cells (Eisenreich et al., J Am. Chem. Soc. 120:9694-9695, 1998) have indicated that the oxygenation reactions of the taxane core are accomplished by cytochrome P450 oxygenases. Thus, for example, the cytochrome P450-mediated hydroxylation (with double-bond migration) of taxadiene to taxadien-5a-ol has been demonstrated with Taxus microsomes (Hefner et al., Chem Biol. 3:479-489, 1996).

Most recently, the taxadien-5a -ol (and acetate ester) have been shown to undergo WO 01/34780 PCT/US00/31254 microsomal P450-catalyzed oxygenation to the level of a pentaol taxadien- 2a,59a, 910p,13a -pentaol) (Hezari et al., Planta Medica 63:291-295, 1997).

Because downstream steps are not yet defined, the above-referenced research summarized in Table 2 involved the pursuit of reactions (the timing and regiochemistry (position) of subsequent taxoid hydroxylations) through the use of surrogate substrates. Thus, labeled (+)-taxusin (the tetraacetate of taxadien- 5,9,10,13-tetraol) was utilized to evaluate hydroxylations at C1, C2 and C7, and the epoxidation at C4/C20 en route to formation of the oxetane D-ring of Taxol.

Microsome preparations from Taxus cuspidata cells, optimized for cytochrome P450-mediated reactions, convert taxusin to the level of an epoxy triol hydroxylation at Cl, C2 and C7 and epoxidation of the C4/C20 double bond of the tetraacetate of taxadien-5,9,10,13-tetraol). Therefore, microsomal P450 reactions have been tentatively demonstrated for all of the relevant positions on the taxane core structure on route to Taxol (Cl, C2, C5, C7, C9, C10 and C13, and the C4/C20 epoxidation), although the exact order for the various positions has not been established firmly.

The screening of the functionally expressed (by CO-difference spectra) clones in yeast (using taxadienol and taxadienyl acetate as test substrates) demonstrated that clone F14 encodes the cytochrome P450 taxane-10 p-hydroxylase.

Similar screening of functionally expressed clones using baculovirus-Spodoptera (especially for clones that do not express well in yeast) also revealed clone F16 as encoding the cytochrome P450 taxane-9a-hydroxylase.

The remaining regiospecific (positionally specific) oxygenases that functionalize the taxane core en route to Taxol can be obtained by identifying additional full-length clones by library screening with the appropriate hybridization probes or by RACE methods as necessary. Each clone can be functionally expressed exhibiting a CO-difference spectrum which indicates proper folding and heme incorporation) in yeast or Spodoptera, as necessary. Each expressed cytochrome P450 clone can be tested for catalytic capability by in vivo (in situ) and in vitro (isolated microsomes) assay with the various taxoid substrates as described below, using GC-MS and NMR methods to identify products and thereby establish WO 01/34780 PCTIUSOO/31 254 the regiochemnistry of hydroxylation of the taxane core. Suitable substrates for use in additional assays are provided in Table 5, below.

WO 01/34780 PCT/US00/31254 Table Substrate Use Taxa-4(20),11(12)-dien (taxadiene) A radiolabeled synthetic substrate employed to search for Taxa-4(20),11(12)-dien-5a-ol and the Radiolabeled synthetic substrates employed corresponding 5a-acetate (taxadienol and to search for early hydroxylation steps and taxadienyl acetate) to assist in sequencing the various regiospecific hydroxylations of the Taxol pathway. These substrates were employed to confirm the taxane 1O-hydroxylase (clone F14) and the taxane 9a-hydroxylase (clone F16), and to indicate the early hydroxylation order as C5, C10 then C9.

Preliminary evidence using these substrates suggests that clones F7, F9, F12 and F51 encode the Cl, C2, C7 and C13 hydroxylases, but the corresponding products (four different diols (and diol monoacetates)) have not been identified and the sequence of oxygenation following 9a-hydroxylation is not yet known.

Taxa-4(20), 11(12)-dien-2a,5a-diol (and Synthetic substrates used to search for the diacetate ester) C1, C7 and C 13 hydroxylases and to assist in ordering the C2, C9 and hydroxylation reactions of the pathway.

Taxa-4(20), 11(12)-dien-5a,9a, 10 13a- Radiolabeled, semisynthetic substrates tetraol and corresponding tetraacetate used to search for the C4/C20 epoxidase (taxusin tetraol and taxusin, respectively) and late-stage oxygenations, including Cl and C7 hydroxylases and the C2 hydroxylase. Also used to assist in ordering the late-stage oxygenation steps of the pathway. Although taxusin (and tetraol) do not reside on the Taxol pathway (Floss et al., Biosynthesis of Taxol, in Suffness Taxol: Science and Applications, CRC Press, Boca Raton, FL, pp. 191-208, 1995), this surrogate substrate is metabolized to the level of a presumptive taxadien-4,20epoxy-1,2,5,7,9,10,13-heptaol (and tetraacetate) by microsomal preparations, but structures of the reaction products have not yet been confirmed by NMR.

WO 01/34780 PCT/USOO/31254 *Taxa-4(20), 11(12)-dien-5a,9a-diol (and Labeled biosynthetic substrates prepared monoacetate and diacetate) from taxadienol (and acetate) using the above-described clones (clone 16). Used in searching for and ordering downstream oxygenation reactions.

*Taxa-4(20),l 1(12)-dien-5a, 10-diol (and Labeled biosynthetic substrates prepared monoacetate and diacetate) from taxadienol (and acetate) using the above-described clones (clone 14). Used in searching for and ordering downstream oxygenation reactions.

Taxa-4(20), 11(12)-dien-5a,9a, 1 OP-triol (an Semisynthetic substrate prepared from acetate esters) taxusin, and used as in above.

Using these natural and surrogate substrates, along with the established expression methods and bioanalytical protocols, it is anticipated that all of the regiospecific cytochrome P450 taxoid oxygenases of the Taxol pathway will be acquired from the extant set of related cytochrome P450s.

Having illustrated and described the principles of the invention in multiple embodiments and examples, it should be apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications coming within the spirit and scope of the following claims.

EDITORIAL NOTE APPLICATION NUMBER 14887/01 The following Sequence Listing pages 1 to 56 are part of the description. The claims pages follow on pages "65" to "68".

WO 01/34780 WO 0134780PCTUSOO/3 1254 SEQUENCE LISTING <110> Croteau, Rodney et al.

<120> CYTOCHROME P450 OXYGENASES AND THEIR USES <130> 56458 <140> (141> <150> 60/165,250 <151> 1999-11-12 <160> 94 <170> Patentln Ver. 2.1 <210> 1 <211> 192 <212> DNA <213> Taxus cuspidata <400> 1 ccatttqgag gaggacqgcg gacatgtcca gqatgggaat acgcaaaagt ggaaatatta ctgttcctcc atcattttgt gaaagcattc agtggttaca ccccaactga ccctcatgaa 120 aggatttgtg ggtatccagt ccctcttqtc cctgtcaagg qatttccaat aaaacttatc 180 gccagatcct ga 192 <210> <211> <212> <2 13> 2 192

DNA

Taxus cuspidata <400> 2 cctttcgqag gaqgcatgcg ctgtttctcc atcattttgt aaactttcag ggaaaccact tccagatctt aa <210> 3 <211> 192 <212> DNA <213> Taxus cuspidata <400> 3 ccattcggag caggagtgcg ctgtttgtcc atcactttgt aaaatttcag gggatccatt ccgagatcat aa <210> 4 <211> 192 <212> DNA <213> Taxus cuspidata <400> 4 ccattcggag caggcgtacg ctctttgtcc atcactttgt aaaatttcag gggatccatt ccgagatcct aa tgtttgtcca gggtgggaat tcgccaagat ggagacatta taaagccttc tctgggttga aggcaattga tccaaatgaa 120 tcctcctctc cctgtcaatg ggcttcccat taaactctat 180 192 catatgtgca ggatgggaat ttgcgaagac agaactatta taaaaacttc agaggttgca ttgtaattga tcctaatqaa 120 ccctccactc cctaccagtg gacaactcat gaaacttatt 180 192 catatgtgca ggatgggaat ttqcaaagac agaactatta taaaaacttc agcqqttgca ttgtaattga tcctagtgaa 120 ccctcctctc cctaccagtg gacaacgcat gaaacttatt 180 192 WO 01/34780 WO 0134780PCT/USOO/31254 <210> <211> 192 <212> DNA <213> Taxus cuspidata <400> cctttcgggg caggcaaacg catatgccca ggatgggagt tcgctaagtt ggagatgtta ctgttcatcc atcattttqt caaaaatttc agcggatacc tcccacttga caccaaggaa 120 aagatctccg gagatccatt ccctcctctc cccaaaaatg gatttcccat taaactattt 180 ccgagaacct aa 192 <210> 6 <211> 201 <212> DNA <213> Taxus cuspidata <400> 6 ccattcggag gaggcgcgcg ctqttcatcc atcattttgt aaaatttcag cagatccatt cccagatctc aatccaattg <210> 7 <211> 192 <212> DNA <213> Taxus cuspidata <400> 7 ccattcggag gaggcctgcg ctgtLtatcc atcattttgt aaaatttcag cagatccatt cccagatctt aa <210> 8 <211> 192 <212> DNA <213> Taxus cuspidata <400> 8 ccgttcggag gcggcctgcg ctgtttatac attattttqt aagatttcag cagatccgct ccaaggtcct aa <210> 9 <211> 192 <212> DNA <213> Taxus cuspidata <400> 9 ccatttggag caggcctgcg ctgtttgtgc atcattttqt aaactctcag ctgatccact tcgagatcct aa <210> <211> 192 <212> DNA <213> Taxus cuspidata cacatqccca ggatgggaat tttcaaagac ggagatatta tagaactttc agcagctacc tcccagttga ciccaacgaa ccctcccctc cctgccaatg ggttctccat aaaacttttt a cacatgtcca ggatqggaat tctcaaagac ggagatatta taaaactttc ggcagctacc tcccagttga ccccaacgaa 120 ccctcctctc cctgccaatg gcttttctat aaaacttttt 180 192 catatgtcca ggatgggaat ttgcaaagac agagatgtta taaaactttc agcagctacg tcccagttga ccccaacgaa cgcttctttc cctgttaatg gattctccgt aaaacttttt cgtatgtcca ggatgggaat tggctaagac ggagatatta taaaacgttc agtagctaca tacctgttqa ccctaaagaa tcctccgctc cctctcaatg ggttttccat taaacttttt WO 01/34780 WO 0134780PCTUSOOI3 1254 <400> ccattcggag gaggcctgcg catctgtcca qqccgggaat ttgcgaaqat ggaqatatta qtgtttatgc atcattttgt taaagctttc agcagcttca ttccagttga ccctaacqaa 120 aaaatttcaa cagatccgct tccttccatc cctgtcaatg gattttccat aaaccttgtt 180 cccagatcct aa 192 <210> 11 <211> 192 <212> DNA <213> Taxus cuspidata <400> 11 ccatttggag gaggcctgcq catctgtqca gqctgggaat ttgcaaagat gqagatatta ctgtttatgc atcattttgt taaaactttc agtcacttca ttccagttga ccccaacgaa 120 aagatttcga gaqatccact gcctcccatc cctqtcaaag gattttccat aaagcctttt 180 cctagatcat aa 192 <210> 12 <211> 192 <212> DNA <213> Taxus cuspidata <400> 12 cccttcggtg gaggccaacg gtcatgtgtg qgatgggaat tttcaaagat ggagatatta ctattcgttc atcattttgt caaaactttt agcagctaca ccccagttga tcccgacgaa 120 aaaatatcag gggatccact ccctcctctt ccttccaagg gattttccat taaactqttt 180 ccgagaccat ag 192 <210> 13 <211> 192 <212> DNA <213> Taxus cuspidata <400> 13 ccatttggag gaggcctgcg cacatgtcca ggatgggaat tttcaaagat tgaaatatta ctgtttgtcc atcatttcgt taaaaatttc agcagctaca ttccagttga tcccaatgaa 120 aaagttttat cagatccact acctcctctc cctgccaatg gattttccat aaaacttttt 180 ccgagatcct aa 192 <210> 14 <211> 192 <212> DNA <213> Taxus cuspidata <400> 14 cccttcgg qaggggagcg cacctgtcca qgatatgaat tttcaaagac tcatatatta ctgttcatcc accaatttgt taaaactttc actggttaca tcccgcttga tccaaacgaa 120 agcatttcgg cgaatccqct cccccctcta cctgccaatg gatttcctgt aaaacttttt 180 ctcaggtcct aa 192 <210> <211> 192 <212> DNA <213> Taxus cuspidata <400> cccttcggcc aqqgtaatcg gatgtqcccc ggaaatqaat tcgcaaggtt ggaaatgqaa ttatttctat atcatttggt tttgagatat gattgggaat taatggaggc ggatgaacgc 120 accaacatgt acttcattcc tcaccctgtg cacagtttgc ctttactact taaacacgtt 180 cctcctacat ga 192 WO 01/34780 WO 0134780PCTUSOO/31 254 <210> 16 <211> 154 <212> DNA <213> Taxus cuspidata <400> 16 ccatttggca agatttgaaa ggagcagctg gaaattgatc tccaatccgt ctctattctc <210> 17 <211> 210 <212> DNA <213> Taxus cuspidata <400> 17 ccgtttggtt cagggagaag tatacgctgq gqaggctgct gacatgacgg agggtttggg aaacctcgcc ttcccttcca <210> 18 <211> 202 <212> DNA <213> Taxus cuspidata <400> 18 cccttcggct gtatccggcg gtgtcggagg gtatcatgtc agaggggttt ggactcacaa cctgcccttt catcztctact <210> 19 <211> 228 <212> DNA <213> Taxus cuspidata <400> 19 ccctttggtg gaggccagcg ctqtcggtgc atcattttgt ataattgcaa gagattccct cctaqatcct attcacttca <210> <211> 219 <212> DNA <213> Taxus cuspidata <400> cccttcggag caggcgtgcg ctgttcttac attattttgt aaagtgttag ggaatccagt cccaggccct cattcgatca <210> 21 <211> 201 <212> DNA <213> Taxus cuspidata <400> 21 ccttttggtq cgggaaggag ttgctctctt tcttcacaac tttgtcacta aattcagatg gtgcgactta ctttcctctt ccttccacag aaaatggttt 120 gagtacacga atga 154 aatqtgtccg ggcatgagtc tggcattgag tgttgttacq gcagagcttc gagtggtctg ttccagaagg tgtgataatc actaacaatg cccaaagcag ttccgttgga gaccattatc tctct actga gggcctctgt tagttcctqa tgaatcgaca gaggactgca ccagcagtcg cgttcctgcg ggtacaacaa ttgacatgag tgcccaaagc gattccgttg gaagccaata taaaacctcq ag ttcatgtcca ggatgggaat tttcaaagat ggagatttta taaaacattc agcaccttca ccccagttga cccagcagaa ctgccctctc ccttccaatg ggttttctgt aaaacttttt cacaggcaac caggtcaaga aaatataa cacctgccca ggatgggaat tttcaaaaac ccagatatta taaaactttc agtggctaca tcccactcga ccctgaCgaa ccctcctctc-cctgccaatg gatttgctat aaaacttttc agqatcaccc atggaataa aggatgccca ggggcaagca tgqccqttgt gacgatggaa WO 01/34780 PCT[USOO/3 1254 cttgcgttgg cacaactcat gcactgcttc cagtggcgca ttgaaggaga gttggatatg 120 agtqaacgct tcgcagcctc cttgcaaaqa aaaqtcgatc tttgtgttct tcctcaatgg 180 aggctaacta gtagcccttg a 201 <210> 22 <211> 63 <212> PRT <213> Taxus cuspidata <400> 22 Pro Phe Gly Gly Gly Arg Arg Thr Cys Pro Gly Trp Giu Tyr Ala Lys 1 5 10 Val Giu Ile Leu Leu Phe Leu His His Phe Val Lys Ala Phe Ser Gly 25 Tyr Thr Pro Thr Asp Pro His Glu Arq Ile Cys Gly Tyr Pro Val Pro 40 Leu Val Pro Val Lys Gly Phe Pro Ile Lys Leu Ile Ala Arg Ser 55 <210> 23 <211> 63 <212> PRT <213> Taxus cuspidata <400> 23 Pro Phe Gly Gly Gly Met Arg Val Cys Pro Gly Trp Giu Phe Ala Lys 1 5 10 Met Glu Thr Leu Leu Phe Leu His His Phe Val Lys Ala Phe Ser Gly 25 Leu Lys Ala Ile Asp Pro Asn Glu Lys Leu Ser Gly Lys Pro Leu Pro 40 Pro Leu Pro Val Asn Gly Leu Pro Ile Lys Leu Tyr Ser Arg Ser 55 <210> 24 <211> 63 <212> PRT <213> Taxus <400> 24 Pro Phe Gly 1 Thr Glu Leu Cys Ile Val Pro Leu Pro cuspidata Ala Gly Val Arg Ile Cys Ala Gly Trp Glu Phe Ala Lys 5 10 Leu Leu Phe Val His His Phe Val Lys Asn Phe Arg Gly 25 Ile Asp Pro Asn Glu Lys Ile Ser Gly Asp Pro Phe Pro 40 Thr Ser Gly Gin Leu Met Lys Leu Ile Pro Arg Ser 55 WOO01/34780 <210> <211> 63 <212> PRT <213> Taxus <400> Pro Phe Gly 1 Thr Glu Leu Cys Ile Val Pro Leu Pro <210> 26 <211> 63 <212> PRT <213> Taxus <400> 26 Leu Ser Gly 1 Leu Glu Met Tyr Leu Pro Pro Leu Pro <210> 27 <211> 66 <212> PRT <213> Taxus <400> 27 Pro Phe Gly 1 Thr Glu Ile Tyr Leu Pro Pro Leu Pro Ser Asn <210> 26 PCT[USOO/3 1254 cuspidata Ala Gly Val Arg Ile Cys Ala Gly Trp Glu Phe Ala Lys 5 10 Leu Leu Phe Val His His Phe Val Lys Asn Phe Ser Gly 25 Ile Asp Pro Ser Glu Lys Ile Ser Gly Asp Pro Phe Pro 40 Thr Ser Gly Gin Arg Met Lys Leu Ile Pro Arg Ser 55 cuspidat a Ala Gly Lys Arg Ilie Cys Pro Gly Trp Giu Phe Ala Lys 5 10 Leu Leu Phe Ile His His Phe Val Lys Asn Phe Ser Gly 25 Leo Asp Thr Lys Giu Lys Ile Ser Gly Asp Pro Phe Pro 40 Lys Asn Gly Phe Pro Ile Lys Leo Phe Pro Arg Thr 55 Cu Spi data Gly Gly Ala Arg Thr Cys Pro Gly Trp Glu Phe Ser Lys 5 10 Leu Leu Phe Ile His His Phe Val Arg Thr Phe Ser Ser 25 Val Asp Ser Asn Giu Lys Ile Ser Ala Asp Pro Phe Pro 40 Ala Asn Gly Phe Ser Ile Lys Leu Phe Pro Arg Ser Gin 55 WO 01/34780 <211> 63 <212> PRT <213> Taxus cuspidata <400> 28 Pro Phe Gly Gly Gly L 1 5 Thr Glu Ile Leu Leu P Tyr Leu Pro Val Asp P Pro Leu Pro Ala Asn G <c210> 29 <211> 63 <212> PRT <213> Taxus cuspidata <400> 29 Pro Phe Gly Gly Gly L 1 5 Thr Giu Met Leu Leu P Tyr Val Pro Val Asp P Ser Phe Pro Val Asn G <210> <211> 63 <212> PRT <213> Taxus cuspidata <400> Pro Phe Gly Ala Gly L 1 5 Thr Glu ile Leu Leu P1 Tyr Ilie Pro Val Asp P.

Pro Leu Pro Leu Asn G.

<210> 31 <211> 63 <212> PRT <213> Taxus cuspidata PCTUSOO/31 254 eu Arg Thr Cys Pro Gly Trp Glu Phe Ser Lys 10 he Ile His His Phe Val Lys Thr Phe Gly Ser 25 ro Asn Glu Lys le Ser Ala Asp Pro Phe Pro 40 ly Phe Ser Ile Lys Leu Phe Pro Arg Ser 55 eu Arg Ile Cys Pro Gly Trp Glu Phe Ala Lys 10 he Ile His Tyr Phe Val Lys Thr Phe Ser Ser 25 ro Asn Glu Lys Ile Ser Ala Asp Pro Leu Ala 40 ly Phe Ser Val Lys Leu Phe Pro Arq Ser 55 eu Axrg Val Cys Pro Gly Trp Glu Leu Ala Lys 10 he Val His His Phe Val Lys Thr Phe Ser Ser 25 ro Lys Glu Lys Leu Ser Ala Asp Pro Leu Pro 40 ]y Phe Ser Ile Lys Leu Phe Ser Arg Ser 55 WO 01/34780 (400> 31 Pro Phe Gly

I

Met Giu Ile Phe Ile Pro Ser Ile Pro <210> 32 <211> 63 (212> PRT <213> Taxus <400> 32 Pro Phe Gly 1 Met Giu Ile Phe Ilie Pro Pro Ile Pro <210> 33 (211> 63 <212> PRT <213> Taxus <400> 33 Pro Phe Gly 1 Met Giu Ile Tyr Thr Pro Pro Leu Pro '210> 34 <211> 63 <212> PRT <213> Taxus <400> 34 Pro Phe Gly 1 PCT/USOO/3 1254 Leu ArqIle Cys Pro Gly Arq Giu Phe Ala Lys 10 Phe Met His His Phe Val Lys Ala Phe Ser Ser Pro Asn Giu Lys Ile Ser Thr Asp Pro Leu Pro 40 Gly Phe Ser Ile Asn Leu Val Pro Arg Ser 55 cuspidata Gly Gly Leu Arq Ile Cys Ala Gly Trp Glu Phe Ala Lys 5 10 Leu Leu Phe Met His His Phe Val Lys Thr Phe Ser His 25 Val Asp Pro Asri Glu Lys Ile Ser Arq Asp Pro Leu Pro 40 Vai Lys Gly Phe Ser Ile Lys Pro Phe Pro Arg Ser 55 cuspidata Gly Gly Gin Arg Ser Cys Val Gly Trp Giu Phe Ser Lys 5 10 Leu Leu Phe Val His His Phe Vai Lys Thr I'he Ser Ser 25 Val Asp Pro Asp Giu Lys Ile Ser Gly Asp Pro Leu Pro 40 Ser Lys Gly Phe Ser Ile Lys Leu Phe Pro Arg Pro 55 cuspidata Gly Giy Leu Arg Thr Cys Pro Gly Trp Giu Phe Ser Lys 5 10 WO 01/34780 Ilie Glu Ile Tyr Ile Pro Pro Leu Pro <210> <211> 63 <212> PRT <213> Taxus <400> Pro Phe Gly 1 Thr His Ile Tyr Ile Pro Pro Leu Pro <210> 36 <211> 63 <212> PRT <213> Taxus <400> 36 Pro Phe Gly 1 Leu Glu Met Giu Leu Met Pro VJal His <210> 37 <211> <212> PRT <213> Taxus <400> 37 His Leu Ala

I

Lys Phe Arg Leu Pro Ser PCTUSOO/3 1254 Phe Val His His Phe Val Lys Asn Phe Ser Ser Pro Asn Glu Lys Val Leu Ser Asp Pro Leu Pro 40 Gly Phe Ser Ile Lys Leu Phe Pro Arg Ser 55 cuspidat a Gly Gly Giu Arg Thr Cys Pro Gly Tyr Giu Phe Ser Lys 5 10 Leu Leu Phe Ile His Gin Phe Val Lys Thr Phe Thr Gly 25 Leu Asp Pro Asn Glu Ser Ile Ser Ala Asn Pro Leu Pro 40 Ala Asn Giy Phe Pro Val Lys Leu Phe Leu Arg Ser 55 cuspidata Gin Gly Asn Arg Met Cys Pro Gly Asn Giu Phe Ala Arg 5 10 Giu Leu Phe Leu Tyr His Leu Val Leu Arg Tyr Asp Trp 25 Glu Ala Asp Giu Ary Thr Asn Met Tyr Phe Ile Pro His 40 Ser Leu Pro Leu Leu Leu Lys His Val Pro Pro Thr 55 cuspidata Arg Phe Glu Ile Ala Leu Phe Leu His Asn Phe Val Thr 5 10 Trp Giu Gin Leu Glu Ile Asp Arg Ala Thr Tyr Phe Pro 25 Thr Giu Asn Gly Phe Pro Ile Arg Lou Tyr Ser Arg Val WO 01/34780 PCT/USOO/3 1254 His Glu <210> 38 <211> 69 <212> PRT <213> Taxus <400> 38 Pro Phe Gly 1 Ser Val Val Ser Val Pro Thr Met Pro Pro Phe His <210> 39 <211> 66 <212> PRT <213> Taxus <400> 39 Leu Arg Leu 1 Glu Asp Cys Ala Gly Thr Lys Ala Ile Leu Tyr cuspiciata Ser Gly Arg Arg Met Cys Pro Gly Met Ser Leu Ala Leu 5 10 Thr Tyr Thr Leu Gly Arg Leu Leu Gin Ser Phe Glu Trp 25 Glu Gly Val Ile Ile Asp Met Thr Glu Gly Leu Gly Leu 40 Lys Ala Val Pro Leu Giu Thr Ile Ile Lys Pro Arg Leu 55 Leu Tyr cuspidata.

Tyr Pro Ala Gly Pro Leu Leu Val Pro Asp Giu Ser Thr 5 10 Ser Val Gly Giy Tyr His Val Pro Xaa Xaa Xaa Val Pro 25 Thr Ile Asp Met Arq Glu Gly Phe Gly Leu Thr Met Pro 40 Pro Leu Glu Ala Asn Ile Lys Pro Arg Leu Pro Phe His 55 <210> <211> <212> PRT <213> Taxus cuspidata <400> Pro Phe Gly Gly Gly Gin Arg Ser Cys Pro Gly Trp Glu Phe Ser Lys 1 5 10 Met Glu Ile Leu Leu Ser Val His His ?he Val Lys Thr Phe Ser Thr 25 WO 01/34780 Phe Thr Pro Val AspE Pro Leu Pro Ser Asn C Ser Leu His Thr Gly <210> 41 <211> 72 <212> PRT <213> Taxus cuspidata <400> 41 Pro Phe Gly Ala Gly 'V 1 5 Thr Gin Ile Leu Leu P Tyr Ile Pro Leu Asp P Pro Leu Pro Ala Asn G Phe Asp Gin Gly Ser P <210> 42 <211> 66 <212> PRT <213> Taxus cuspidata <400> 42 Pro Phe Gly Ala Gly A 1 5 Val Thr Met Giu Leu A Arq Ile Glu Gly Glu L Gin Arg Lys Val Asp L Ser Pro <210> 43 <211> 1455 <212> DNA <213> Taxus cuspidata <400> 43 PCTIUSOO/31254 ~ro Ala Giu Ile Ile Ala Arg Asp Ser Leu Cys 40 ;iy Phe Ser Val Lys Leu Phe Pro Arg Ser Tyr 55 ~sn Gin Val Lys Lys Ile 70 ral Arg Thr Cys Pro Gly Trp Glu Phe Ser Lys 10 he Leu His Tyr Phe Val Lys Thr Phe Ser Gly 25 ro Asp Glu Lys Val Leu Gly Asn Pro Val Pro 40 ly Phe Ala Ile Lys Leu Phe Pro Arg Pro Ser 55 'ro Met Giu rg Arg Gly Cys Pro Gly Ala Ser Met Ala Vai 10 la Leu Ala Gin Leu Met His Cys Phe Gin Trp 25 eu Asp Met Ser Giu Arg Phe Ala Ala Ser Leu 40 eu Cys Val Leu Pro Gin Trp Arg Leu Thr Ser 55 WO 01/34780 atggatacct attcttggca cttccccctg acacctcgga ctaattggtc aacgaggaaa gattgcgtta gccttgggcc catatcaacc ctcgtcttct cgacttcatc ccaggaactc tctgtaatag ctactgtcgg atactqqata accttgatat cagctggaaa gatatgaaat ggatatattc tggagaatat gaagaattca ataccatttg ttactgttta gaaaagattt tttcctagat PCT[USOO/31254 tcattcagca caattcttct gaaacatggg agtttatcga atcccgcagt agctggtgcg tggggaaaac cccaggcgt t aaaaatggaa ccatttcaac atcttttgga gttttcgtaa aaaggagaag tgttgqtcac acttctcttt ttaagctgct tacttggcaa atacatggca gcgaggcttt tatgttcacc gaccttcaag gaggaggcct tgcatcattt cgagagatcc cataa cgagtcttcc tttgatatta cttccctctc cgacagaqtg tgtaatatgc gatqtctttg cggagtggag gcagaattat ggggaaagat cagcttgttt aactgtagct agcactttac aagcgatctt cttcaaagat tctacttcac ctcctctagt taaaaaggat agcagttcag gacagatatt tcatactacg attcgaggat gcgcatctgt tgttaaaact actgcctccc ccacttcttc agtggtaaac attggggaga aagaaattcg ggctcctccg cccaacgcag catgggattg gtgqccaaaa gaggtgaagg ttcggtataa atgggacttg gcgcggtcga cgttcaggag gaaagaggga gccttatacg cctgaatgct agagaggaaa gaaactttga gactatgatg catagtaaag caaggaaggc gcaggctggg ttcagtcact atccctgtca ttztctcttac agtacagatc ctatagcact gcctggtttt caaaccgttt tactgaaact tacgtaccgc tgagttcaga tgcttcctct acgatgagca tgagtattcc agctcgatqa cagct tcaag attcattcqc acaccacaat atgagaatat tcagctggaa ggatgttccc gctatacaat aggagtattt at g tggc tcc aatttgcaaa tcattccagt aaggattttc tctcgctgtt ttctcgtaaa tatatcagat caagacttcg cctcctctcc cttggggcag actagcccgc gatcgaacac gataagaagc ccaacaqaag cctagacttt aattatqtct cgaccaagat agacaaggag ttcaccactc agctcaagag ggatctgaag tccagtttat accaaaagga cgatgagccg ttacacattc gatggagata tgaccccaac cataaagcct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1455 <210> 44 <211> 1455 <212> DNA <213> Taxus cuspidata <400> 44 atgcttatcq accctcacac cttccccctg acacctgata ttaattgggc aacgaggaaa gattctgttc tgtttgggcc catatcaacc cttgtcttct cgacttcatc ccaggaacta tctgtaatag ctattgtcg atcctggata acgttggtat caattggaaa gatatgaaat ggaaattttc tggaggattt gagaaattca ataccattcg ttagtgt tta gaaaaaattt gttcccagat <210> <211> 1506 <212> DNA aaatggatac ttat'tcttct gaaacatggg aatttttcgg atcccacaat aactggtgcg tggggaaaat cccaagcgct aaaaatggaa ocat cgcaac atcttctgqa catttcgtaa aaagqagaag tgttgctcac acttctcttt ttaagctggt ttcttcgcaa atacgtggca gcaaggcttt tatgttcacc gaccttcaag gaggaqqcct tgcatcattt caacagatcc cctaa cttcgttcag ttttatattc cttccctctc cgatagaatg tgtgctctgc gatgtttccg aggagaggag gcagaattac gggaaaaggt cagcttattt aacagttgtt agcacttcac aaacgatctg cttcaaagat tctacttcat gtcctccaat taaaaaggat aqcagttcag gacagatatt ttatactaca attcgaagag gcgcatctgt tgttaaagct gcttccttcc ctcgagtctt tgtagtaaac attggggaga aagaaattcg ggt tcctccg cccaactcat catcggattg gtgtccaaaa gaagtgaaga tttggtatta acqggacttt gcgcggtcga cgtttaggcg gaaagaqgga gccttatacg cctgaatqct ggagaagata gaaaccttga cattatgatg catagtaaag caaggaaggg ccaggccggg ttcagcagct atccctgtca cccctgttct aatacagatc cgataqcact gcaaggtttt gaaaccgttt ccagcaaact tacgtaccgc tgagttcaga tqcttcctct ccgatgagca tgtqtattcc agctcgatga cagcttcaag atccattcgc acaccacaat acgaaaatat tcagctgggc ggatgtqtcc gctatacaat aggagtattt atgtggctcc aatttgcqaa tcattccagt atggattttc tctttccctt ctctcttaaa ggcat cacag caagacttcg ictcctctcc cctggggcag actagcccgc gatccaacgt gataaqaagc acaacaagaa gctcgacttt gattatgtct cgaccaagat tgacaaggag ttcaccactc agctcaagag ggatctgaag tccagtttac cccaaaagga tgacgacccg ttacacattc gatggagata tgaccctaac cataaacctt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1455 WO 01/34780 WO 0134780PCTUSOO/3 1254 <213> Taxus cuspidata <400> atggattcct gatcaqtctt cttgtgttgc ggcttccctt catatqtttt gggcatccca cacaaqctgg atcgtqacca gggcctcatg aataagcatt ttctccattt aaaactcttt tctaattttc at cgaaagca tcggtgctgc gacaactttt atatttaagc ggaatacttg aaatatacat tttcgcaagg gctatggtga ttcaagcctt ggggcaggca atccatcatt tccggagatc acctaa tcagttttct ccagtactgc tctttcgatt tcattggaga ttgatgagag cagctgtgtt tqcagtcqtc aaacaggaga ccttacagag ggaagggtaa caagcagctt tagaaactat gtaaagctct gaagaaaaga tcgccttcaa cttttatgct tgctctccgc gcaataaaaa ggcaagcagc tcatcgccga caaattacag caaqatttgg tacgcatatg ttgtcaaaaa cattccctcc aaaaagcatg tcttctgtcc taaaagccgg gacga tacag attgaagaaa ctgcgggcct tgggcccaac ggagcaccgc ttatacgct agatgaagtg gttttttgat tcttgtggga tcgggcgcgt tatgcgttct agatgaaaga t cacgcctca caatccagaa ggacqgtgaa tcaagaaaca tattcatcat tacaagtagg ggatgqaaag cccaggatgg tttcagcgga tctccccaaa gaagcgaaat ctcgcattca ccctctacta ttcttgcggg tttgggcgtg gcgggaaacc tccttcgtca atctttcttg aaaatgagtt aacatqcttc attaatgatg actttqtcgg tccaagctgg gggatagctt gggaatccat tacgacacca tgctatgaaa gaaatgtgtt atgaggcttt gatggctata aaagaagagt tatgtggctc gagttcgcta tacctcccac aatggatttc tcggccaagt cagctgctgt atttccctcc cacttcgatc tattcaagac ggtttattta aactggttgg gtgtcctgaa ccaaaatcca cttcgataag aggatcaaca ttcccctcga atgaaattct ctaccagtaa tgacggacac ccgtttcgCC aagtagttca ggaacgatct tccctccagc taattcccaa acttcgatga cgtacacatt agttggagat ttgacaccaa ccattaaact catacaccg tgccattttt aggaaatttt agaatcgcct gtca tta act c tcga at gag gcagcaatcc cgagtttctg ggagaatatc acagctcgtc gqaacaactt cattccagga gtctcgttta aaatctactg ggagatcctc cacagtttgt agaacaat tg gaaagctatg gtttggatca aggatggaaa acdagacaat cttacctttc gttactgttc ggaaaagat t atttccgaga 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1506 <210> 46 <211> 1503 <212> DNA <213> Taxus cuspidata <400> 46 atggatgccc caggcttccc ctcgtcatca cctttcattg tttgtggqg cccactataa ctgttgc:acg gtgaaaaggg gcagggctac aaatggaagg aattcagcta atattcjaaaa tatcgcaaag aagagaaaag ttgctcagct tgttttgcaa aagatgttgt gcgtcaaata acatggcaag aagaccatga tggacaactt ccttcgagat ggaggacgCc catcattttg gggtatccag tttctcttgt ctgctattct cttctaaacg gcqagacttt aaagggaggg tactctgcgg tgtcgtggtc gagatgatca agctttacat gaaaagatqa tct tgt tt tt tcattcttgc cactcaaggg acgaactgcg tcagagatga tgctggatgc cttccaatcc aaaaggaggg tgctccagga atgacattaa attctacaca tcgaagagga ggacatgtcc tgaaagcatt tccctcttgt aaacagcaca gtccactgcc ccgttcctct agagttcgtg qaaatttgqa ccctgcggqja cgcccaaatt ccgcgttctg aggtaaaatg agtgaatgta caatatatac ctcacatttc gagcttgaag ctcaagatta aagagggaaa ctcctatgac agaatgcttt agaagaaatc aagtctacgg tcacgatggt tcagaaagac agatgggcat aggatgggaa cagtggttac ccctgtcaag qttgcaaaat ctcactgcta cttaaacttc aaggctcttc cgtqtgttca aaccgcttag gccaqaatcc cgtgtcgcac agtgcactta ctgagtttgg gataaagagc ggcatacctt cggaaaaaaa qcgtctagca ccactgagcg accaccactt gaaaaagtag acaatgaagg atqctttctc tacacaattc atatatttca ttggatgctt tacgcaaaag accccaactg ggatttccaa ttaatgaggt t tgcaggcat ctcctqqaaa gatcagacac agacttcatt ttctttccaa tgggtctcaa tagcaggttt tcagaaatca taagagatct gaaagcaaca taaacattcc ttctctccgc atcaagatct acgaggcagt cacaaatgac ttcaagagca atatcaaagc caqtatttg caaaaggatg agcagccaga atacattcgt tggaaatatt accctcatga taaaacttat aacgcagcta tattgtgctc actaggcctc acttcgacaa gcttgggaag cgaggaaaaa ttctgttgca tttgggctct tatcaatgaa tgtcatggac actgcatgaa cggatttctg tttactggaa tctctctgtt cttagacaac tctgatttta attggagata catgaaatac aacacttcgt gcaggttgta taaattcatg accat ttgga actgttcctc aaggatttgt cgccagatcc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 01/34780 PCTfUSOO/31254 t ga 1503 <210> 47 <211> 1476 <212> DNA <213> Taxus cuspidata <400> 47 a tggatacca a tttccgncg cgacgatcct atacagttct aagaaattcg gggccggcgg cccaagtctt catcgcatct attgggaaaa gaggtgaagg ttcaatataa gtgggaagta gcacgggcga agatcgggcc gaaaaaggga gcctcgtatg cctgaatgct ggagaagaag gaacccctaa agttgqaagg gqgagaqaag aagcccctgg tgggaatttg ggttgcatta accagtggac tacgagcaag ctctggcagc atgtgaatct tgggggcact gtaaggtctt gaaaccgctt tcctgaaact tacgttcggc tgaattcaga tgcttccttt atqatgacag tgactattcc agctggacga taaattctgg atccactgac acactactqt atgaaaaagt tcaat tggaa gnatgccccn ctatacaatt agttcttcaa atccttacac caaaggctga taattgatcc aactcatgaa ttttggcgaa ttttcttggt cccccctgga tcagtcagaa caagacttct agttctqtcg gtttgggqag tctgggtcqa aat gcaacqfl ggttagaggc acaacgtgag tctgaacatt aattcttttt taatcaagat agacaaggag ttcaccaacg tgttcaagaq ggatctgaaa ccagcttttg ccaaaaggat tgaaccagac attcatacca actattactg gaatgaaaaa acttattccg gttattcagc attgttattt aatttaggtt aaaccccata ctaattgggg aacgaagaca qattccgttg t ttctgggtc catttcqatg ctcattttct caactccatg ccaggaactc gctttqatag cttctgtcgt atcctcgaca gtcttga tat cagttgggaa gctatgccat gaatgtttcg gggcaattat aaattcaagc ttcggagcag tttgtccatc atttcagyggg agatca cagagtattc tctcgatctt tacctttcat catttttcga at cccacggt agctggtgca cqgccaaaag cc catg ctt t acaaatggaa ccattgctac gtctgctgga tttttcgtaa agaacagaag ccttgctcac acttctctgt tgaagcttct tacttqctag atacatqgca aaqaqctttc gtggccaflct cttccagatt gggtacgcat cctttgttaa atccat Lccc tcctctcatc cagttccact tggcgagacg tcjagagagtg ggtactctgc gtccgcaggg agaagagagc acagaat tat ggqaaaagat ctccctgttc tacaatact t agctgtcaag aagagagctg cttcaaagat tatgcttcat cgcctccaat taaaaaggag agcaattcag cctgatattc tatagtcaat cgaggaagga atgtgcagga aaacttcagc tccactccct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1476 <210> 48 <211> 1503 <212> DNA <213> Taxus cuspidata <400> 48 atggatagct cagtcttccc ct ctqgttct ttccctttca acgttttttg catcccacag aaactggtgc ctgtccaaaa ccccaagctt caaaaatqga aacattgcaa catcttttgg cgttttcgta qagagcagaa gtqttgctca aatttttcat cttaagcttc atagttggca tatacatgca cgcaaggcta ttctqgtcac tcaatttctt ctgctgttct tccttcataa ttggggagac atgagagggt ttgtactctg agqcgtcatt gaggggagga tgcagggtta agggaaatga gcagcctgtt aagccattgt aagctcttga qaagqgatct ccttcaaaga ttatgcttca tcttctctag ataaaaagga aggt tgtgca ncacctatat cttatactac gaqaggcat t ttccctttcc aaacggttcc cataccattc gaaqaaattc cgggcctgag gcccaactct gcatcgcata tgttgctaaa tgaagtgaag tttcggcata tctgggaagt tgcgcggtct gcgtttqggc tgaaagaggg tgcctcgtat tcctqattgc gggagaagaa ggaaagtatg ccattatgat acacgggaaa ggagcagatt ctgatcacaa tctgttactc ttgagggcac gqtgttgtat ggaaaccgct tccgagaaac ttacgtgctg atgagttcag gtgcttcctc aatgatgaac ctgtctgttc aagctggato acggcttctg aatccactca gataccactg tatgaaaaac atcagctqqa aggatgctcc gggtatacaa gaagaatact ttgggggatt ctattcttgg t cccccctgg ttcgatcaga tcaagactcq ttcttctctc taattqggaa cacttgcccg aaatccaaca tgataagaac accaacagga cgctcgactt agattctttc agaatcaaga cagacaagga tttcaccaac taqttcaaga acgatctgaa ctccagtttt ttccaaaagg tcaatgaagc cattcagttc cgttctactt aaatttaggc aacacctcag gatagttgg caacgaggac atattccatt ctttttgcga tcatatcaag cctgatcttc acagcttcat tccagqaact ttctttaatg tcttctttct aatcttcgac qqqtttgatg acaattggga agctatgaaa tggatcgtat atggaatata qgacaagttc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 WO 01/34780 WO 0134780PCTfUSOO/31254 atqccttcga gattcgagga gcaggtctgc qcgtatgtcc catcatttta ttacaacttt ggggatccat ttcctcctct taa <210> 49 <211> 1452 <212> DNA <213> Taxus cuspidata aggcaaatat gttgctcctt acacattctt gccattcgga aggatgggaa tttgcaaaga ccqagatatt actgttcgtc cagcagctac atcccaattg accccaaaga taaaatttca gcctaccaat ggattttcca tqaaactttt taccagatct 1320 1380 1440 1500 1503 <400> 49 atggatacct tttctggqcg ccacctggaa cctcagaagt attggqcatc qaggaaaagc tcccttctgg ttgggccccc atcaaccaaa gtcttctcca cttcatcttc ggatctggtt ttaatgaaaa ctgtcggtgt ctcgacaact ttgattttta ttagaaatac atgaaatata aattttcgca agggttttat gaattcagac ccgttcggag ctgtttatac aagatttcag ccaaggt cct taattcagat ttgttgtgct atttaggctt ttttaaacga ccacagttgt tggtgcggat ggaaaacggg aagagttgca aatggaaggg ttgcaaccag ttttggaaac ttcgtaaagc tcagaagaaq tgctcacctt tctctgttct agctcatgtc tttcccatag cttggcaagc aggctttgac gttcgccttt cttcaagatt gcggccztgcg attattttgt cagatccgct aa ccagtcttcc tttqatattc gcccttcatt gagggggaag tctctgcggc gtctttgccc acaggaacat gaatcatgtg qaatgatgaa cttqtttttc tat tgtaatg gcttcaggca cgatctgcgt caaaqatgaa acttcatggc ctccaatact agagaaggga cattcaggaa tgatattcat taccacgcac cgaggggcaa catatq Loca taaaactttc cgcttctttc cctgatttcc cgttataaac ggggagacaa a aa t ttggtc tcctcgggaa aactcataca cggattgtgc gccaaqatga gtgaaggtgc ggtataaacg ggagctgtgt cggtcggagc tcaggcgcag agaggaaatc ttatatgaca gaatgctacg gaggagatcg accttgagaa tacgatggct agcaatgaag ggaaagaatg ggatgggaat agcagctacg cctgttaatg tttcctttac accgatccqc taacatttgc ctgttttcaa accgt tt t ct tgaaactcct gtaccgcact gttcagacat ttcctctgat atgagcacca gtattccgct tcgatggaat cttcaagcaa cattgacaga ccacaatttc agaatgtagt gttggaagga tgttccctcc atacaatccc aatattttaa tgccttctta t t gcaa aga c tcccagttga gattctccgt tctcacagcg tctcaaactt atctcaacct gacgtcgcta cctctccaac ggggcaggat aggacgtttt tcagcatcac aaggaacctt acaggagcga cgcct ttcca tctcatttct ccaagatcta caaggagatc accactcac ccaagagcaa tctgaaatct ggtttacgga aaaagggtgg tgagccagat cacattcata agagatgtta ccccaacgaa aaaacttttt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1452 <210> <211> 1512 <212> DNA <213> Taxus cuspidata <400> atggacgct t ggctcttaca ttgctcatgc ttccctttga cagttttttq gaacgcacgg aaggtggtgg gccaacacaa cccgggtcgt gagaaatgga tccgttgcaa ccatcaatct ggaactagtt ctgatagaaa gtctccgtgt ctcgataatt ttgacattca ttaatatttt ccgatcgtat tccgttggaa tcggcgaaac atqagaggca tagtactctg agtcatcgtg acggagagaa tacagaatta agggaaaaga ccgccttgtt t gcggaaact atcggagagc ggagaagaag tgctcacctt tctccgggct agctcatgtc aaagggccct tctttccatt aagccagtct attacaattg aaaaaaattt cggtccgtct gccgagcgct gcatcggatc tgtggggaaa tgaagtgaag tttcggtgtn gcactttgcg tctggaggca cgatctqcqc caaagacgaa acttcacgca ctcctctgct gctgcaaaac acagtcgttg tctgtgaagc ttgagggcat gqttgtgttt ggaaaccqtt ttcatcaaac ttacqcgccq atgaggtcag gtgctcgatt aatgacgagg ggcagttt tt cggttgaagc tcgggcttgg ggaggaaatc tcgtatgaca gaatgctatg ttaatggagt ccttcattac ttcccccggg ttcgatctaa tcaagacatc tagtgctcgc tcatcgqaga cactgcttag aaatcgaaca tggtaagaaa aaagaaaaag ctattccgct tggataaaat catctggtaa ctctgacaga ccacaacttc acaaagtagt cgtgcagctc tattctcctg gaactttggc cacgactcaa actagtcgga caaccagaac ggattccatt atatcttggt tcatatcaat gaatgtcttc gatccgacct ggactttcca cctctcttct tgaggatctq caaggagatc agcactcacc tcaagagcaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 WOO01/34780 ctgagaatag atgaaatata tcatttcgga aaagttttat aaattcaggc cccttcqaag ctgttcatcc agcatttcgq caaaggtcct PCT/USOO/3 1254 tttccaataa catggcaagt agaccatcgc gggcaactta ct tcgagatt qaggggagcg accaatttgt cgaatccgct aa aaaggaggga ggtgcagqaa cgacattcag taccacacat cgaagaggga cacctgtcca taaaactttc cccccctcta gaagaaatca act ctgagga tacgatggct ggqagagatg ggaaagcatg ggatatgaat actggttaca cctgccaatg gct tgaaaga tgttccctcc atacaattcc agtatttcag tggctcctta tttcaaagac tcccgct Iga gatttcctgt tctgaaagac gcttttcgga aaaaggatgg tgagccccaa cacattcttg tcatatatta tccaaacgaa aaaacttttt 1080 1140 1200 1260 1320 1380 1440 1500 1512 <210> 51 <211> 1494 <212> DNA <213> Taxus cuspidata <400> 51 atggatagct cctgctattc cqttacaatc ggggagacca gatagattga gtactctgcg atggaagggc ggcgaggatc caaaattatc gg ta aaga tg accctgtttt actattcttg qggcttcagg agagatctgc ttcagagatg atgtttcatg tact ccaat c aaaaaggaag gcagttcaaq acigatatto tatactacac tttgaggatg cgcacatgtc gttaaaaatt ctacctcctc tcatttttct tttcccttac accgatcctc tacaattatt agaaattcgg ggcctgcggg ccaagtcttt atcgcatctt tgggtagaat aagtgaaggt tcgatgtaaa tgggaagttt cgcgqctgaa gttcaggcat aaaaagqgaa cttcatatga ctgaatacca gggaagaaat aatcactacg attatgatgg atctgagaga aaggcaggca cagqatggga t cagcagcta tccctgccaa gagaagcata cctcgcacct tgttaaactt gcggacactc tcctgtttac aaacaaatta catgaaactg acgcactgca gagttcagaa gcttcctttg tgatggacac gtcagtcccg gct tgatqaa agcttctgat ctcactgaca caccactgtt t gaaaaagta cagttggaag aatgtaccca gtatacaatt agagtacttc tgtgactcct attttcaaag cattccagtt tggattttcc ggaacaaaat attctcgcca ccccctggaa cgatcagaaa atgacttccc gttctttcga attggggaag cttgctcggt ataggacacc gtaagagggc caacagaagc ctggactttc attctctcct gatcaagatc gaccagggga gcaccaatgg tttcaagagc gatttgaaat ccagtttttg ccaaaaggat cctgagcctg tacacatatg attgaaatat gat cccaatg ataaaacttt ttgggcagct ttattcttct agttaggttt cacctcaaaa taattgggca acgaggacaa attccattgt ttttgggcgc atttcaatga ttatcttctc aacttcatca caggaactcg ctctaataaa tactqtcggt ttctggacaa ccttgatatt agttggaaat ctatgaaata qaatatttcq ggagggtttt aagaattcag taccatttgg tactqtttgt aaaaagtttt ttccgagatc ggagtcttcc cttgctcttc tcctctcatc gttttttgat tcccacagtt gctggtagag tgctaaaaga tcaagcttta aaaatggaag cat tgcaagc tcttctgqaa ttatcgtaaa acgcagaaga gttgctcacc cttttctgct taagcttcta aattggcaat tacatggcaa taaggctatc atgttcacct gcct tcaaga aggaggcctg ccatcatttc atcagatcca ctaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1494 <210> 52 <211> 1524 <212> DNA <213> Taxus cuspidata <400> 52 atggaaacta attctcggcg cttccacctg ct tcgatcac ttcaagactt ttagttctgt ctgatggggc gcactagccc agaaccgaac ttgataagag caccaacagg ccgctggact gaaattctct aatttgggca cccttqttct gaaagttagg aaacacctca cactaattgg ccaacgagga aggactccct ggtttttaqg atcatatgaa agctcatctt agcgacttca ttccaggaac cctctttaat acttatgcag tctccatctc tttccccgtc aaagtttttc aaatcccct a caagcttgtg cctggccaaa cccccaagct tgaaaaatgq ctccaatgca tcatcttttq tcgcttacgt aaaaagcaga ctngagtttc t tccqtcata attggggaga qatqatagag gtggtcatgt cagttqgaag agacaagagg ctacanaatt aagggaaaag agcagcttgt gaagctgttg aaagcccttc agaaaagatc ttccctttat gaaaccgatc cgatacagtt tgcagaaatt gcgggcctgc cgcccaattc accaccgcac atatgactaa atgaagtgag tt t tcga tat ttgttggaag aggcgcgatc ttgtttcagg cctcacacct ctctgttaaa cctgagggca tggtggtgtt gggaaaccgg cttgatgaaa cttacgtgct aatcagttca gacgcttcct caatgatgag tatgtctatt taagctggat gatagcttct WO 01/34780 gatgatcaag accgacaaag gtttccccaa gtagttcaag aaggatttga cctccacttt attccgaaag ttcaatgaac tacacattca acggagatat gactccaacg ataaaacttt tttcccagat PCTIUSOO/3 1254 atctactgtc agatcctcga tggttttgac aqcaattggg aagcca tgaa ttggatcatt gatggatgat cgttgaaatt taccattcgg tactgttcat aaaaaatttc cagcagatcc ctcaatccaa ggtgttgctc caacttttct attgaagctc aatagttqcc atacacatgg tcgcaaggct tttatggaca taggccttca aggaggcgcg ccat cat ttt agcagatcca attcctccc ttga accttcaaag cttctgcttc ctctcctcca aataaaagga caagtagttc atggttgata acttacggta agatttgaag cgcacatgcc gttagaactt ttccctcccc ctccctqcca acgagagagg atgcctcgta atccagaatg taggagaaga aggaaacact ttgattatga cacaccigag aagacgggcg caggatggga tcaqcaqcta tccctgccaa atgggttctc aaatccactg tgacaccact ctatgaaaaa aatcagctgg gagaatgttc tggctacaca agaagagtac tgtgactcct attttcaaag cctcccagtt tqggttctcc cataaaactt 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1524 <210> 53 <211> 1539 <212> DNA <213> Taxus cuspidata <400> 53 atggcttatc gccgcggtgc aacaatggaa cagttgggaa atgctcatga gaagttctga tacataqcgt atgaagaaaa gtaaqagagg gcggtcgccg atcttttcca tcggaggtgt tggatggatt gtcattacga ccaaaagaca a tggaaa a! a actacgt tgq caagaagaga agtatggaat ttgcttatcc agaaccagaa gcgctggcat gaqtttttcg gccgttgtga gaaggagaqt tgtgttcttc cggagttgct ttacaatttt gaagattgcc agcttcccaa aattgggttc aaactcatga ataattacaa tatgcgtggt aagaggtgtc tcaatctgag gtaacgatga ctgagacggc tgcagggtat aaattataga taattgacgc tcaaagccgt aatgggcgat tcgaatccgt acctacaatg cgcacgaatc ttctcgttaa tcaaaccaaa atatggttcc cgatggaaca tggatatqag cccaatggag cgaaaattta attcttgttg ccccggccca ccgtaatctg cgttcctqcc tctggttttc ggatatagtt ggaattgttg tgttataat t caagacgct g cggcgggaa t gggagctttt acagcggcgc gcaacaccag cctgttgcag cgttttgggt gagcgcgat g tgtgggaaga tgtggtgaaa gacccaagat cgcgtgggcg aagatttttg ctttggtgcg tgcgttggca tgaacgcttg gctaactagt tcqggagacc gggattttct attccatqgc gaagagctcg gttatcgttt gccagccgac ttctctccct aatgccagaa cgttcggtgt tcatccct.La agcaqcgtca aacattggag a tgacgaagg aggacgagag atggagaaca atttttctgg cttgaaaacc aagagggtgg aagacgat ga Lgcactgtca ataggaaaag ggcanaaatg ggaaqgaaag caactcatgc gcagcctccq agcccctga gagctcaatc acatactgcg cgatcgtggg caaagaaaca cttcctctgc ccgaaagcgc acggacctta gaatcoagtc gggagaagag cacagggact ccgccattaa at tatttt cc cacacgatta cgatggagga ccgatggcgt gcggagcgga ctgaggtggc tgaaagaaat gattatatcc atggatactt atccaaacgt tggact tgca gatgcccagg actgcttcca tgcaaaaaaa tccagcaata cgggctgaga aaatctccac cggacccatc catggcaaaa cgcaggaaaa ctggagacag gttgagatcc caagcagggt catgttgcaq agaaatgatg atgqatqgac tttcgaccag cactcaacaa caccatcaca gacgacgtcc caagaaagtg qatctgggaa gqcggtgcct cattcctgaa gtgggatgat aaaaggaaaa ggcaagcatg gtggcgcatt agtcgatctt 120 180 240 300 360 420 460 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1539 <210> 54 <211> 1530 <212> DNA <213> Taxus cuspidata <400> 54 at ggatgt ct attcttttca cgttctaaac ggcgagtcgt gagagagtga gttctctgcg atgtcgtggc ggcgaaggcc tttatccgtt ttgtcctcag gccgttcctc tactgttcct agaatttcqg gcctgcagg ccaaatcctc atatgatcat aaaaagtaca tgctgttgct tgttggacta gaaggctctt gaatgtcttc aaaccggcta tatgaaactc ccgctccgca qtagcaaaat ggcattgttc cccccaggga cgatcaaaCa aagacgtcat at cctggcga atgggggaga ctgcaaggct ttaacgaatq tgcccctgct aattaggtta cagttgaaca taattgggca acgaggagaa agtctattac ttttcagccc tttccctgct gctgttccta ccctttcatt atttttggac tccgacagta gctggtgcag tgccaaaagg tggtgctctg WO 01/34780 cagaaataca ggaaacgacc tgtttgttct attatagctg gcacttcaag atgqatctga t tcaa agat g ctgcttcatg tcttccaatc aaattggaag gtcqttcagg actgacattc tacacaacac ttcgatcagg cqttcatgtc gttaaaacat ctctgccCtC cacacaggca PCT[USOO/3 1254 taggccaaat aagtgagtgt tcaatataaa tcggagtttt cacggtcgaa gctcaggat t acagaggaaa gatcctatga ccgaatgcta gagacgaaat aaacgttacg attataatgg atcccaagga aagqgaaact caggatggga tcagcacctt tcccttccaa accaggtcaa gagtaaaaca agttgctttg tgagaagcat ggctgttccg gcttaatgca agcgactagc tccatgcagc caccactgtt tgaaaaagta cacatggaaa attgtatccg ttacataatt aatgtatttc tgtagctcct attttcaaag caccccagtt tgggttttct gaaaatataa atagaaaatc gtaggagatc gaacqggaac gtggatcttc attctctCCg aatcaggatc gatgaggaaa tcagcaatgg gttcaagagc gatgtgaaat tcaatttttg ccaaaagggt agtgagccgg tacacatttt atggagattt gacccagcag gtaaaacttt atattaatga tcgtcttcga gactgtttga ccgggtttgc gtttgataga ttctttctgt tcctcgacaa cctgcgtttt aattgqggat ccatgaaata gatcatttcg ggaagctttt agaaattcct taccctttgg tactgtcggt aaataattgc ttcctagatc gaaatggaag tatttcggcc gcttttggag ttaccatcgg aaagagaaaa gtttctcac cttttccggg taagcttttg actttcgaat tacatggcaa ccaggccatc gtggacacca gccttcgagg tggaggccag gcatcatttt aagagattcc ctattcactt 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1530 <210> <211> 1545 <212> DNA <213> Taxus cuspidata <400> 55 atggcattcg atacagcgcc ccttcatggc attctatctt ccagctttgg gccttcgctt ttcagtatgg atcctctctg ctcattcgtt aqqctctctg ggacctgttu tctgtgttct gatcttcagg cagaaattgg ttaattgatg gatqttgtga accatcgaat caggagctcg cegaaazat t ttagttcctc acgcgactga accqtgt tcg gagtttgaat ttgagtgttg gaaggtatga& ttggagacca aagcagctac gtagaattag ccgttattgg cgctttcgga ttattgcctc ctcgcccacg ctccttacgg caaccagaat cgttgtttga atctcacgtt attccgagga tacttggagc gtttcatagc tgattgatca ttctcatctc aagccaccgc gggcattggc acacgcatat tgcaggcaat acgaagccat ttgtgaatgc atcctgaacg tgattccgtt ttacgtatac taattgacat ttatcaaacc tgttatictt aaggcacaaa gaatctt cat gagctatqga ttcagatctg tctgtctgca ttcctactgg tgactccttc cagttgccag tagtatcatc atacgaagaa atttgaggtt tqctatgaaa ccgtgagaag tgcaacagac ccttacaatg ggctctgatg cggacgcagc tgtgaaagaa tgaggattgc ttgggcaatt gtttttgaag tggttcaggg gctggggag gacggaaggt tcgcct tccc ttcactctgq t tgcagggga ctgcttacac ccaatcatgc gcgaaagaat ggaaagcatg cgaaaccttc agacacatcc cgagagqaca ctccgtatgg gcggatcatt ggagatttcc aaactgcagc agagqgagag aaccatgaaa ctgaacgcag cagcaccctc cgattactag acqttgaggc actgttggag cacagagacc agcggaaaag aqaagaatgt ctgctgcaga ttgggactca ttccatctct ctgccctgtt aggtgaaggc agaaagtgcc atcttcaact gcttcacaac taggatatga ggaaaatgtg gcgtagagga ctccagtcaa ttgccaacaa ttaaccagat tgccgtttct agaaaagaga tcgatgcaaa ttcagtccga gtacagatac atattttgag aggaagcaga tatatccagc ggtaccatgt cggcagtgtg aggttgacgt gtccgggcat gcttcgagtg caatgcccaa actga gctagtcgtc accacaacct tat tcaccga cggtctccga aaatgacaaa ctacaaaatc cacgatccag agtttctgct catgaaagcg gaaattatca gataaaacag caagtggctt tgtctttatg tgcacaagac tagtaacgac atcctcgqtg caaagcccag tctgcacgag cgcacctctc ctccgcagga ggaacggccg aaaaqqgcgg gagtctggca gtctgttcca agcagttccg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1545 <210> 56 <211> 484 'z212>' PRT <213> Taxus cuspidata <400> 56 Met Asp Thr Phe Ile Gin His Giu Ser Ser Pro Leu Leu Leu Ser Leu 1 5 10 WO 01/34780 Thr Leu Ala Lys Gin Tyr Pro Leu Ile Phe Ilie Asp Leu le Gly Phe Leu Leu Ala Val Leu 115 Val Glu His 130 Gin Ala Leu 145 His Ile Asn Leu Ile Arg Ile Asn Asp 195 Val Ala Met 210 Phe Arg Lys 225 Ser Val Ile Ser Asp Gin Gly Asn Ser 275 Leu His Ala 290 Lys Leu Leu 305 Gin Len Glu Lys Asp Leu Ile Asn Th r Phe Ser Ser Gly 125 Al a Giu Lys Le u Leu 205 Pro Glu Gi y Lys Phe 285 Th r Ile Glu Val PCT/USOO/3 1254 Ser Giy Gly Phe Arg Lys Thr Ser Asn Arg Pro Asn Thr Giy Gly Pro Giu His 160 Leu Pro 175 Phe Giy Giu Thr Thr Arg Met Ser 240 Ala Ser 255 Glu Arg Phe Leu Ile Phe Gin Glu 320 Ser Trp 335 Glu Thr WO 01/34780 WO 0134780PCT/USOO/3 1254 '-210> 57 (211> 484 <212> PRT <213> Taxus <400> 57 Met Len Ile 1 Leu Len Ser Lys Gin Tyr Pro Len Ile Phe Phe Gly Leu Ile Gly Phe Leu Leu Ser Ser Ser 115 Gin Glu His cuspidata Gin Met Asp Leu Thr Leu Arg Ser Ser Gly Gin Thr Asp Arg Met His Pro Thr Ser Asn Giu 100 Lys Len Len Arg Ile Val WO 01/34780 130 PCTIUSOOI3 1254 Gin 145 His Leu Ile Val1 Phe 225 Se r Ser Gi y Leu Lys 305 Gin Al a Le u Asp Cys 385 Giu Pro Arg Ly s Met Ser Ser Giu Ile Gin Val1 Ser His Phe 220 Asp Leu Phe Asn Leu 300 Asn Gi u Aia Arg Gly 380 Tyr Giy Arg Met As n 4 WO 01/34780 Thr Asp Pro 465 Vai Pro Arg PCTUSOOI31254 Leu Pro Ser Ile Pro Val Asn Gly Phe Ser Ile Asn Leu 470 475 480 Ser <210> 58 <211> 501 <212> PRT <213> Taxus cuspidata <400> 58 Met Asp Ser Phe Ser Phe 1 Val Ile His Arg Asp Gin Phe Thr Ala Ala Val Ala Ser Arg Pro Ser Thr Asn Ile Giy Glu Thr Ilie Gin His Met Phe Phe Asp Giu Thr Ser Leu Thr Gly His 100 Asn Arg Phe Ile Tyr Ser 115 Pro Asn Ser Phe Val Lys 130 Thr Gly Giu Glu His Arg 145 150 Gly Pro His Ala Leu Gin 165 Gin Giu Asn Ilie Asn Lys 180 Leu Pro Ser Ile Arg Gin 195 Phe Asp Ilie Asn Asp Giu 210 Giu Thr Ilie Leu Val Giy 225 230 Ser Asn Phe Arg Lys Ala Leu Arg Ala 245 WO 01/34780 Leu Ser Arg Ala Ser Thr 275 Giu Arg Gly 290 Phe Met Leu 305 Ile Phe Lys Gin Giu Gin Cys Trp Asn 355 Giu Thr Met 370 Ile Ala Asp 385 Ala Met Val Glu Pro Asp Ala Pro Tyr 435 Gly Trp Giu 450 Val Lys Asn 465 Ser Gly Asp Leu Phe Pro <210> 59 <211> 500 <212> PRT <213> Taxus <400> 59 Met Asp Ala 1 Val Thr Gin PCTIUSOO/3 1254 Gly Ile Lys Asp Phe Ser Val Cys 320 Val Val 335 Giu Met Ala Gin Lys Val Trp Lys 400 Phe Asp 415 Tyr Val Cys Pro His Phe Lys Ile 480 Ile Lys 495 cuspidata Leu Ser Leu Val Asn Ser Thr Val Ala Lys Phe Asn Giu 5 10 Leu Gin Ala Ser Pro Ala Ile Leu Ser Thr Ala Leu Thr 25 WO 01/34780 Ala Ile Al Ser Ser LE Glu Thr LE Phe Val Gi Leu Leu G1 Leu Val LE 11 Gin Ile Al 130 Asp Asp Hi 145 Ala Gly LE His Ile As Leu Vai Ar 1s Ile Tyr As 210 Ile Leu Ai 225 Tyr Arg L Aia Leu Le Ser Asn GI 2'7 Gly Lys Pr 290 Leu Asp Ai 305 Lys Met Le Gin Leu Gi Gly Lys Glu Glu Lys 100 Se r Arg Arg Gin Glu 180 Asp Ly s Ser Aila Glu 260 Asp Leu Ser Se r Ile 340 Thr Leu Asp Val Pro Val Ala 140 Gi y Ala Vali Ile Glu 220 Ile Lys Se r Phe Asn 300 Met Lys Glu Lys Phe Leu Lys Gi y 110 Trp Lys Leu Ile ValI 190 Phe Leu Gly Ile Leu 270 Asp Phe Leu Vali Ile 350 PCTUSOO/3 1254 Arg Arg Ile Gly Arg Gin Thr Ser Asn Arg Ser Ala Arg Giy Gly Ser 160 Arg Asn 175 Leu Ser Phe Asn Lys Ile Phe Leu 240 Leu Ser 255 Ala Ser Giu Arg Ala Met Ile Leu 320 Gin Glu 335 Thr Met WO 01/34780 Lys Asp Ile 355 Leu Arg Met 370 Asp Ile Asn 385 Trp Thr Thr Asp Lys Phe Ala Tyr Thr 435 Trp Giu Tyr 450 Lys Ala Phe 465 Gly Tyr Pro Ile Ala Arg <210> <211> 492 <212> PRT <213> Taxus <400> Met Asp Thr 1 Ser Pro Leu Ile Phe Ser Pro Gly Asn Gly Ala Leu Lys Lys Phe Val Vai Leu Asp Lys Leu 115 PCTIUSOO/3 1254 Glu Ser Met Asn Val Val 400 Gin Pro 415 Leu Asp Pro Gly Phe Val Ile Cys 480 Lys Leu 495 cuspidata Ile Arg Ala Ile Ile Ser Ile Phe Ser Leu Gly Leu Gin Ser Giu Gly Lys Vai Cys Gly Pro 100 Val Gin Ser WO 01/34780 Gly Gin Asp 130 Arg Ser Ala 145 Ile Gly Lys Lys Gly Lys Phe Ser Ile 195 Arg Giu Gin 210 Thr Ile Pro 225 Ala Arg Ala Arg Arg Glu Ser Ser Leu 275 Lys Gin Ile 290 Thr Thr Val 305 Pro Glu Cys Ser Lys Lys Pro Tyr Thr 355 Leu Leu Glu 370 Ile Gin Phe 385 Gly Arg Giu Phe Giu Glu Ala Gly Val 435 Ser Leu Met Asp 180 Al a Leu Leu Lys Len 260 Len Len Ser T yr Gin 340 T rp C ys Gin Gin Gly 420 A rg Aia Arg 150 Se r Val Ser Gly Ile 230 Asp Ser Phe Asn Th r 310 Lys Gin Al a Glu Asp 390 Phe Pro Cys Lys Arg Leu Gly Met Gin Vai Len 185 Phe Phe 200 Len Asp Gly Thr Ile Len Leu Asn 265 Asp Gin 280 Ser Val Le Ilie Vai Gin Val Asn 345 Gin Glu 360 Len Ser Gin Len Giu Pro Asp Pro 425 Gly Trp 440 Gin Pro Arg 170 Pro Asn Thr Len Phe 250 Ser L ys Met Len Giu 330 Trp Pro Len Cys Asp 410 Tyr Gi u Se r 140 Al a Phe Val1 As n Leu 220 Arg Len As n Asn His 300 Len Leu Asp Xaa Phe 380 Xaa Phe Phe Al a Arg Gin Asp Gly 190 Asp Giy Al a Gin Asp 270 Len Se r Ala Ile Lys 350 Pro Trp Ile Pro Pro 430 Ala PCTIUSOO/31 254 Ile Len Asn Tyr 160 Lys Trp 175 Len Ile Arg Gin Ser Met Val Lys 240 Asn Arg 255 Len Len Thr Asp Tyr Asp Ser Asn 320 Len Ala 335 Ala Met Xaa Gin Lys Ala Val Asn 400 Ser Arg 415 Phe Gly Gin Len Len Len Phe Val His Pro Phe Val Lys Asn Phe Ser Gly Cys Ile Ile WO 01/34780 450 Ile Asp Pro 465 Thr Ser Gly PCTIUSOO/3 1254 455 460 Asn Giu Lys Ile Ser Gly Asp Pro Phe Pro Pro Leu Pro 470 475 480 Gin Leu Met Lys Leu Ile Pro Arg Ser 485 490 <210> 61 <211> 500 <21.2> PRT <213> Taxus cuspidata <400> 61 Met Asp Ser Phe Asn Phe Leu Ser Leu Pro Leu Val1 Thr Giu Ile 135 Leu Tyr T rp Ile Gin 215 Le u 225 230 Arg Phe Arg Lys Ala Leu Asp Ala Arg WO 01/34780 Le u Phe 285 Asn Th r Lys Giu Val 365 Arg Gi y Tyr Lys Val1 445 His Asp PCTIUSOO/31254 255 Thr Ala Asp Glu Ser Phe Leu Met 320 Val Gin 335 Ile Ser Gin Giu Ala Xaa Asn Ile 400 Asn Glu 415 Val Ala Pro Gly Phe Ile Ile Ser 480 Lys Leu 495 Pro Leu Pro Thr Asn Gly [The Ser Met 490 Phe Thr Arg <210> 62 <211> 483 <212> PRT <213> Taxus <400> 62 Met Asp Thr 1 Thr Leu Thr cuspidata Leu Ile Gin Ile Gin Ser Ser Pro Asp Phe Leu Ser Phe 5 10 Ala Phe Leu Gly Val Val Val Leu Leu Ile Phe Arg Tyr WO 01/34780 Lys His Arg Phe Ile Gly Leu Asn Giu Ile GMy His Leu Leu Ser Tyr Met Lys 115 Glu His Arg 130 Giu Leu Gin 145 Ile Asn Gin Ile Arg Asn Asn Asp Glu 195 Val Met Gly 210 Arg Lys Ala 225 Leu Met Lys Asn Gin Asp Asn Pro Leu 275 Hi-'s Gly Leu 290 Leu Met Ser 305 Leu Glu Ilie Asp Leu Lys PCT[USOO/3 1254 Ser Ala Leu Lys Leu Pro Pro Giy Asn Leu Gly Leu Pro WO 01/34780 Arg Met Phe 355 Ile His Tyr 370 Ser Pro Phe 385 Glu Phe Arg Tyr Thr Phe Glu Phe Ala 435 Thr Phe Ser 450 Asp Pro Leu 465 Pro Arg Ser <210> 63 <211> 503 <212> PRT <213> Taxus <400> 63 Met Asp Ala 1 Val Val Gin Val Ala Phe Gin Ser Ser Gly Glu Thr Gin Phe Phe Ser Leu Val Arg Leu Val 115 Ser Ala Phe 130 PCTUSOO/3 1254 Thr Asp Leu Cys Pro Asp 400 Pro Ser 415 Gly Trp Val Lys Ser Ala Leu Phe 480 cuspidata Phe Asn Ilie Leu Gly Ser Ile Thr Ilie Val Lys Leu Leu Gin Leu Asp Giu Arg Gly Glu Arg 100 Leu Ala Asn Ile Lys Leu WO 01/34780 Gly Gin L, 145 Pro Gly SE His His I' Asp Len Vi 14 Gly Vai A 210 Arg Lys LU 225 Gly Thr S4 Ilie Leu Si Leu Ala Si 2' Asp Gin G.

290 Ser Gly L, 305 Leu Thr P! Vai Gin G Ilie Ser L 3 Gin Glu TI 370 Thr Ilie A 385 Lys Val L.

Ser Gin P His Val A

A

Ile 150 Asn Lys As n Gln Ala 230 Arg Ile Gin Pro Ala 310 Met Arg Leu Met Gin 390 Thr Phe Thr Ala Gi y Gi y 185 Se r Arg Phe Gin Arg 265 Val1 Asp Asp Ser Ser 345 Met Pro Gly Thr Ser 425 Pro Tyr Gin Lys 190 Le u Ser Asp Len Arg 270 Th r Asp Ala Asp Gi y 350 Gin Phe Lys Gin Gly 430 Giu PCTIUSOO/3 1254 Len Giy i160 Ile Gin 175 Val Len Phe Phe Ile Len Phe Pro 240 Asp Lys 255 Ser Giy Phe Lys Asn Phe Leu Thr 320 Lys Vai 335 Gin Gin Val Vai Arg Lys Gly Trp 400 Tyr Phe 415 Gly Lys Arg Thr Cys Pro Gly Tyr Gin Phe Ser Lys Thr His Ile Len Len Phe Ile His WO 01/34780 Gin Phe Val 465 Ser Ile Ser Val Lys Leu PCTUSOO/31254 Thr Gly Tyr Ile Pro Leu Asp Pro Asn Glu 475 480 Leo Pro Pro Leo Pro Ala Asn Gly Phe Pro 490 495 Ser <210> 64 <211> 497 <212> PRT <213> Taxus cuspidata <400> 64 Met Asp Ser Phe Ile Phe 1 Leu Glu Ser Ser Pro Ala Ala Ile le Leu Leu Leu Lys Leu Pro Pro Gly Lys Gln Leu Leu Arg Thr Leo Asp Arg Leo Lys Lys Phe His Pro Thr Val Val Leo 100 Ser Asn Gin Asp Lys Leu 115 Lys Leu Ile Gly Gin Asp 130 Arg Ile Leo Arg Thr Ala 145 150 Gin Asn Tyr Leo Gly Arg 165 Gin Lys Trp Lys Gly Lys 180 Gly Le Ile Phe Ser Ile 195 Gly His Gin Gin Lys Gin 210 Gly Ser Leu Ser Val Pro Leo Asp Phe Pro Giy Thr Arg Tyr Arg WO 01/34780 Gly Leu Gin PCTJUSOO/31254 Leu Ile Ala Arg 245 Leu Lys Leu Asp Glu Ile Leu Ser Ser 250 255 Arg Asp Val Leu Gly Ilie Thr Val 310 Giu Tyr 325 Lys Lys Tyr Thr Phe Giy Thr Ile 390 Leu Arg 405 Phe Giu Giy Gly Ile Leu ?ro Val 470 Asp Asn His Leu Leu 335 Asp Arg Ilie Ser Giu 415 Tyr Gi u As n Asp Leu Pro Pro Leu Pro Ala Asn Gly Phe Ser Ile Lys Leu Phe Pro Arg <210> <211> 507 <212> PRT <213> Taxus cuspidata <400> Met Giu Thr Lys ?he Giy Gin Leu Met Gin Leu Giu Phe Leu Pro Phe 1 5 10 WO 01/34780 Ile Leu Thr His Arq Asn Pro Val Ilie Thr Pro Gin Phe Lys Thr Ala Gly Asn Glu Ala Pro 115 Ala Lys Arg 130 Phe Leu Gly 145 Arg Thr Glu Arg Thr Leu Len Phe Phe 195 Leu Leu C-lu 210 Pro Gly Thr 225 Giu lie Leu Gly Ile Ala Lys Asp Giu 275 Phe Ser Leu 290 Val Leu Thr 305 Val Val Gin Gin Ile Ser PCT/USOO/3 1254 Phe Arg Giy Phe Ser Gin Gly Val Gly Pro Gin Leu Leu Leu Ala Arg Ser Ser 160 Glu V~al 175 Ser Ser His His Asp Phe Leu Asp 240 Val Ser 255 Thr Phe Asp Asn Pro Met Glu Lys 320 Gly Glu 335 Gin Val WO 01/34780 WO 0134780PCTIUSOO/3 1254 345 Pro Asp Gly Pro Pro 425 Th r Tyr Pro Pro Gin 505 Asn Thr 25 Asn Gly Leu Pro Thr 105 T yr Pro Leu Gly Tyr Thr His 395 Ser Arg 410 Phe Gly Glu Ile Leu Pro Leu Pro 475 Pro Leu 490 Ser Asn Leu Ser 10 Ile Leu Asn Gly Asn Leu Ala Lys Ala Val 90 His Asp Ile Ala Gi y 365 Ile Arg Glu Gi y Leu 445 Asp Asn Ala Asp Le u Arg Gin His ValI Val1 Asn 350 Ser Phe Pro Lys Glu Glu Giu Asp 415 Ala Arg 430 Phe Ile Ser Asn Gly Phe Asn Gly 495 Arq Ala Leu Gly Leu Pro Leu Giy Gly Pro Ser Ser Phe Ala 110 Tyr Lys <210> 66 <211> 512 <212> PRT <213> Taxus <400> 66 Met Ala Tyr 1 Ser Pro Ala Phe Tyr Ile Gly Pro Ilie Leu Pro Asn Met Leu Met Ala Met Ala Arq Pro Glu cuspidata.

Pro Glu Leu 5 Ile Ala Ala Leu Arg Gly Pro Trp Pro Arg Asn Leu 70 Lys Leu Gly Lys Glu Val 100 Ser Ala Ala WO 01/34780 WO 0134780PCTUSOO/31254 Ile Cys 145 Val1 Ser Le u Gly Glu 225 T rp Tyr Arg Leu Lys 305 Thr Al a Val Val His 385 Arg Val1 Asn Tyr Leu 150 Val1 Val Met Thr Phe 230 Gly Ile Thr Thr cay 310 Ala Glu Ile Arg Asp 390 Val1 Le u Lys Gly 135 Asn Ser Al a Leu Al a 215 As n Ile Thr Gin Asp 295 Ile Met Glu Trp Leu ~375 Cys Asn Al a Gly Pro Tyr Trp Arg Gin Met Lys Lys Ile Al a Val1 Val1 Gin 200 Ile Ile Gin Lys Gin 280 GI y ?he Ser Ile Giu 360 Tyr Thr Ala Phe Lys 440 Arg Ile As n 185 Ile Lys Gly Arg Ile 265 Pro Val1 Leu Ala Giu 345 Ser Pro Val1 Trp Lys 425 Glu 140 Giu Ser Lys Ser Met 220 Phe Thr Gin Ile Th r 300 Ala Glu Val1 Tyr Pro 380 Tyr Gly Arg Leu T rp Leu 190 Asp Glu Trp Ala Gin 270 Asp Glu Thr Pro Arg 350 Gin Leu Ile Asp Leu 430 Ser 160 Lys Se r Gly Ser Asp 240 Asp Thr Le u Ile Ser 320 Val1 Arg Val1 Pro Giu 400 Asn Xaa Phe Phe Asp Met Val Pro Phe 445 WO 01/34780 Gly Ala Gly 450 Met Glu His 465 Giu Gly Glu Lys Val Asp PCTUSOO/3 1254 Arg Lys Gly Cys Pro Gly Ala Ser Met Ala Val Val Thr 455 460 Ala Leu Ala Gin Leu Met His Cys Phe Gln Trp Arg Ile 47 0 475 480 Leu Asp Met Ser Giu Arg Leu Ala Ala Ser Val Gln Lys 485 490 495 Leu Cys Val Leu Pro Gin Trp Arg Leu Thr Ser Ser Pro 500 505 510 <210> 67 <211> 509 <212> PRT <213> Taxus cuspidata <400> 67 Met Asp Val Phe Tyr Pro 1 Cys Phe Pro Ala Ile Leu Val Leu Pro Leu Leu Leu Gly Leu Pro Pro Gly Lys Leu Phe Leu Lys Ala Leu Giu Arg Val Lys Asn Phe His Pro Thr Val Val Leu 100 Ala Asn Glu Giu Lys Leu 115 Lys Leu Met Giy Giu Lys 130 Met ile Ilie Arg Ser Ala 145 150 Gin Lys Tyr Ile Gly Gin 165 Glu Lys Trp Lys Gly Asn 180 Asp Leu Val Phe Asp Ilie WO 01/34780 Lys His G.

210 Gly Val D~ 225 Ala Leu G' Giu Lys Aj Asp Leu L( Cys Ser A.- 290 Ser Tyr A 305 Ser Ser A! Ile Leu S( Lys Ser Mf Tyr Pro S( 370 Tyr Asn G- 385 Tyr Thr T1 Leu Pro S( Phe Leu Pj 4: Ser Lys M( 450 Ser Thr Pt 465 Leu Cys Pj Ser Tyr S( <210> 68 <211> 514 PCTUSOO/31254 Ala Val.

His Arg 240 Leu Ile 255 Asn Gin Asn Pro His Gly Leu Leu 320 Leu Gly 335 Asp Val Arg Leu Ile His Thr Pro 400 Lys Phe 41i5 Tyr Thr Glu Phe Thr Phe Asp Ser 480 Pro Arg 495 WO 01/34780 PCTIUSOO/3 1254 <212> PRT <2 13> Taxus cuspidata <400> 68 Met Ala Phe Giu Ala Ala Thr Val Ile Leu Phe Thr Leu Ala Ala Leu 1 5 10 Leu Leu Val Val Ile Gln Arg Arg Ar Ile Arg Arg His Lys Leu Gln 25 Gly Lys Val Lys Ala Pro Gin Pro Pro Ser Trp Pro Val Ile Gly Asn 40 Leu His Leu Leu Thr Gin Lys Val Pro Ile His Arg Ile Leu Ser Ser 55 Leu Ser Giu Ser Tyr Giy Pro le Met His Leu Gin Leu Giy Leu Arg 70 75 Pro Ala Leu Val Ile Ala Ser Ser Asp Leu Ala Lys Giu Cys Phe Thr 90 Thr Asn Asp Lys Ala Phe Ala Ser Arg Pro Arg Leu Ser Ala Gly Lys 100 105 110 His Val Gly Tyr Asp Tyr Lys Ile Phe Ser Met Ala Pro Tyr Gly Ser 115 120 125 Tyr Trp Arg Asri Leu Arg Lys Met Cys Thr Ile Gin Ile Leu Ser Ala 130 135 140 Thr Arg Ile Asp Ser Phe Arg His Ilie Arg Val Giu Giu Val Ser Ala 145 150 155 160 Leu Ile Arg Ser Leu Phe Asp Ser Cys Gin Arq Giu Asp Thr Pro Val 165 170 175 Asn Met Lys Ala Arg Leu Ser Asp Leu Thr Phe Ser Ile Ile Leu Arg 180 185 190 Met Val Ala Asn Lys Lys Leu Ser Gly Pro Val Tyr Ser Glu Glu Tyr 195 200 205 Glu Glu Ala Asp His Phe Asn Gin Met Ilie Lys Gin Ser Vai Phe Leu 210 215 220 Leu Gly Ala Phe Giu Val Gly Asp Phe Leu Pro Phe Leu Lys Trp Leu 225 230 235 240 Asp Leu Gin Gly Phe lie Ala Ala Met Lys Lys Leu Gin Gin Lys Arg 245 250 255 Asp Val Phe Met Gin Lys Leu Vai Ile Asp His Arg Giu Lys Arg Gly 260 265 270 Arg Val Asp Ala Asn Ala Gin Asp Leu Ile Asp Val Leu Ile Ser Ala 275 280 285 Thr Asp Asn His Glu Ilie Gin Ser Asp Ser Asn Asp Asp Val Vai Lys 290 295 300 WO 01/34780 PCT/US00/31254 Ala Thr Ala Leu Thr Met Leu Asn Ala Gly Thr Asp Thr Ser Ser Val 305 310 315 320 Thr lie Glu Trp Ala Leu Ala Ala Leu Met Gin His Pro His Ile Leu 325 330 335 Ser Lys Ala Gin Gin Glu Leu Asp Thr His Ile Gly Arg Ser Arg Leu 340 345 350 Leu Glu Glu Ala Asp Leu His Glu Leu Lys Tyr Leu Gin Ala Ile Val 355 360 365 Lys Glu Thr Leu Arg Leu Tyr Pro Ala Ala Pro Leu Leu Val Pro His 370 375 380 Glu Ala Ile Glu Asp Cys Thr Val Gly Gly Tyr His Val Ser Ala Gly 385 390 395 400 Thr Arg Leu Ile Val Asn Ala Trp Ala Ile His Arg Asp Pro Ala Val 405 410 415 Trp Glu Arg Pro Thr Val Phe Asp Pro Glu Arg Phe Leu Lys Ser Gly 420 425 430 Lys Glu Val Asp Val Lys Gly Arg Glu Phe Glu Leu Ile Pro Phe Gly 435 440 445 Ser Gly Arg Arg Met Cys Pro Gly Met Ser Leu Ala Leu Ser Val Val 450 455 460 Thr Tyr Thr Leu Gly Arg Leu Leu Gin Ser Phe Glu Trp Ser Val Pro 465 470 475 480 Glu Gly Met Ile Ile Asp Met Thr Glu Gly Leu Gly Leu Thr Met Pro 485 490 495 Lys Ala Val Pro Leu Glu Thr Ile 11e Lys Pro Arg Leu Pro Phe'His 500 505 510 Leu Tyr <210> 69 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 69 tcggtgattg taacggaaga gc 22 <210> <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer WO 01/34780 <400> ctggcttttc caacggagca tgag <210> 71 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 71 attgtttctc agcccgcgca gtatg <210> 72 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 72 tcggtttcta tgacggaagc gatg <210> 73 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 73 attaaccctc actaaacctt ttgg <210> 74 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 74 attaaccctc actaaacctt tcgg <210> <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> PCT/US00/31254 24 24 24 24 WO 01/34780 attaaccctc actaaaccat ttgg <210> 76 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 76 attaaccctc actaaaccat tcgg <210> 77 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 77 attaaccctc actaaaccgt ttgg <210> 78 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 78 attaaccctc actaaaccgt tcgg <210> 79 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> 79 attaaccctc actaaaccct ttgg <210> <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR Primer <400> attaaccctc actaaaccct tcgg PCT/US00/31254 24 24 24 24 24 24 WO 01/34780 WO 0134780PCTUSOO/3 1254 <210> 81 <211> 1539 <212> DNA <213> Taxus cuspidata <400> 81 atggacgctt ggctcttact cttctcctgg cttggctacc ccgcaacagt attggggacc gaaaacaaqc tccattgctg ctgggacccg atgaatgaga gtcttctcca cttcatgacc ggaacaaatt ctgatagaaa ctctctgtgt ctcgacaact ttgacattaa ctgagaa tag augaaatata tcatticgta aaagttt tat aaattcatgc cccttcggag ctgttcttac aaagtqt tag cccaggccct ttaatgtttt ctgaaaatct tgttgatccq ctttcatcgg tct t tg at ga gcacagtggt tggtggaggc ggaaaaacgg gagcattaca aatggaaggg tcgcaaccag ttttggaaac atcggaaagc ggagaagaag ggctcacttt tctccacctt agctcatqtc tttccaacaa catggcaagt aggccatcac agacaactta cttcaagatt caggcgtgcg attattttgt ggaatccagt cattcgatca aatgggccct ttccqttaca ttccaaaccc cgaaacatta gaggcagaag gctgtgcggt atcctggccg agagaagcat gaattatatg gaaagagcaa cttgtttttC cgcacttgcg ccttgaagcg cgat ctgcga caaagacgaa gcttcatgca ctcctctact aaaggaggga tgtgcaggaa tgacattcat tagtacacat cgaagaggaa cacctgccca taaaactttc ccctcctctc aggatcccc ctagcaaaat attaccgtca caatcttgtg caattgttgc aaatttgggt ccctcaggaa agttcttcca cqqatcttac gcgaagauga gtgaaggtgc ggtgtcaatg ggtgtttttt cggttaaaac tcaggcgtgg gaagggaatc tcatatgaca gaatgctatc gaagaaatca act ctgagga tatgatqqtt gggagagaag ggaaggcatq ggatgggaat agtggctaca cctgccaatg atggaataa ttgataattt cagcgattgC taaaccttcc aggcatttcg ctgttttcaa accgtttgct ttaaattgat gcgccgcggt ggtcagaaat ttcctttggt atgacggaga ctattccact tggataaagt catctggtaa ctctgacaga ccacaacct c a caa a gtag t gcttgaaaga tgttccctcc atacaat ccc agtatttcaa ttgctcctta tttcaaaaac tcccactcga gatttgctat catgcagctc cgtcattact tccgggaaag atcgaacagg qacttcacta gctctccaac cggagaggat aaaccgttac cgaacat cat aaaagagaat acgggaacqg ggattttcca cctttcttct tgaggat ctg caaggagatc agcactcacc tcaagagcaa tctgaaagac gctttttgga aaaaggatgg tgaaccagag cacattttta ccagatatta ccctgacgaa aaaacttttc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1539 <210> 82 <211> 1458 <212> DNA <213> Taxus cuspidata <400> 82 atggatgccc atggcaggca ggaaatctag agcactccac tccataatcg tcgaacgaga gaagattqtc cggtttttg cgccacatca qacctcgtct gagcaacttc attcccggat acccatttga qatttqctct gagatcctcg cttaccatgt gagcaatttg aaggagatqa tttggaacat tiaagcaatt ttatcctctt gcttccctct agcagtttat gcatcccac acaagctggt tcggcggcaa gtcctcaagc atgaaaaatg tctccgtcgc ataacttqtt tcagttacca tagaaaagag ctgttttgct acaacttttc tgattaaagt gaatactctc aatattcatg ttcgcaaagc ggaagtttc cttccgctct ggttggggag tgaagagaga agtagtgctg gcagatgtca aacggqagag attgcagaat gaagggaaag aagccgcttg qqaaqttatt taaagcgatt gagaaatgag cactttcact tatgttactt cttggcctcc caccaaaatg gcaagttgtt cat cactgac ccttccattc aaacgccatt acactgcagt atgagcaaat tgtggacctg tggccgagct cagcatcgga cat ttcgcta gatgaggcca ttttttggta cttgtgggat caggcaaggg ctgcqtgcag gacga aaggg catqgatcat catccagaaa gagggagaag caggaaacat attcattaca ttttcgttac cctctgtaaa tcgtgaggtc ttgqggatgt ccggaaaccg ccatgatgaa tcgtacgcgc aaatgagctc ctgtacttcc t aactgagga ctttttctgt ccaccctcgc gcactgcatc ggaattcact atgactccac qctatgaaaa aaattqcttq tgcgcatgta atggttatac cctcgcagta actcccccct act tggetcg gttcaagact gttggttctg actcatcggc tgcactaact gggaatccaa tttggtaaaa gcacctqCag tccactcaac tqacatcatg tgagaatcaa ggcggacaag caattcccca agtggctcaa gaaagacctg tcctcccatt aattccaaaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 WO 01/34780 ggatggaaac gccgat caat tacttacctt acattactgt aatgaaaaac ctctattcca PCT/USOO/31254 ttttatggac tcaagccatc t cggaggagg ttctccatca tttcagggaa gatcttaa aacttacagt actcaaacca aggaagagta tttcaaggac aaqatttgag gaggaaggga agcatgtaac cccttacaca catgcgtgtt tgtccagggt gggaattcgc caagatqgaq ttttgttaaa qrcttctctq ggttgaaggc aattgatcca accacttcct cctctccctg tcaatgggct tcccattaaa 1200 1260 1320 1380 1440 1458 <210> 83 <211> 1482 <212> DNA <213> Taxus cuspidata <400> 83 atqqacagct gagtacattc aaatcctctc caattcttgc aaattcggtc qcggcaggga agctctttaa cgcattttgc gctaaaatga gtgaagacgc gatataaatg ggaagtatgg cgttcgaagc tctggcaaag agaggaaatc tcgta tgata gaatgctatg gaggaaatca acactgagga tat gacggtt gtgaaagaag ggaaggcatg ggatgggaat ggcagctacc cctgccaatg tcactt ttgt tatcccttac ataaacttcc gttcacttcg ctgttttcaa gccgattagt agaagctaat gttctgcatt qtacagaaat ttcctttgat a tgagcccca ctgttcgCct tggatqaaqc cttcaagtaa cactgagaga ccactatttc acaaagtagt gttggaagg3 tgttccctcC acacaattcc agtacttcaa tggctcctta tctcaaagac tcccagttga gcttttctat aaccatcaaa cctcacagct ccctggaaac atcacaaaca gacct cgcta tctgtctaac ggqggagaat atcccgcttt cqagcgtcat aagagggctc acaggagCga cgactt tcca tctccattct tcaagatctt cgaggagatc accaatggtt t caagagcaa tctgaaagct actttttgga aaaaggatgg tgaacctggc cacat tctta ggagatatta ccccaacgaa aaaacttttt atgggaaaaa attcttctct ttgggcttcc cctqaatttt attggqgCtC gaggacaagc tccattctgt t tgggtCCCC atcaacgaaa gtcttctcca cttcatcatc ggaactcgct ttaataaaaa ctttcggtgC ctcgacaatt ttgacattga tttggaatac atgaaatata tcattccgca atcqttttat aaattcaggc ccattcggaq ctgtttatc aaaatttcag cccagatctt tttggcaagt tcttcttccg cttt tat tgg tttttqacga ccacagtgat tggtqcagat ataaaagaga aagctttgca aatggaaggg ttgcaaqcag at ttggaaag ttcgtaaagc gcagacgaag tgctcagctt tttctcztat agctgctgtc ttgccaataa catggcaagt aggctatggt ggacaactta cttcaagatt gaggcctgcg atcattttgt cagatccatt aa cattcaggtg ttacagaaac ggagaccata gagggtgaag attctgcggg ggaatcgcca agaggaacac aacttacatt aaaagaagaa tctgtttttc tcttgttgca cgttgaggcg cgatctgctt caaagatgaa acttcatgcc ctccaatcca aaaagaggga agtgcaggaa tgatattaat cagtaca cat cgagcatgat cacatgtcca taaaactttc ccctcctctc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1482 <210> 84 <211> 1491 <212> DNA <213> Taxus cuspidata <400> 84 atggaactgt gtqqgcatgg ttgaagacgg tggggagaat tacgacacac actgtgqtca tttctcaaca tcgcagggcg aaccctgaaa tggcatggcg gctgccgatt ttcagtgatt gggaaggcga cataggactt ggaatatqtt catccgcatt ccagaagaaa ctctqqgcta ggaaggccaa tqttqggtcc gttggcccaa cagaacacaa ctagcgtggg gccaaatcat ttttcatagg tcagcgcggg aacgagcgcg cca tgcacaa tctgccatgg cgctataatt tatgcctccg tctcggctca acacggcaaa ggatgccaac atctctcaac aaggatgcgq aagattcgaa ccaagcctac gttaaagccc gcttttatct cgccgccatg aagtggagag atctccattg tatgctcctc attcctccag tggaataacc attttcacaa aggttcatcc gctctcatcg cgaattatac ggactggtgt cgccaagt ta ggaaaagaat caccctctcg gtcactcaga gaggggggaa caacagcaac ttttgctgtc gaagcatqggg agagcaaccc cccacattct tcat taacga gaaagcacgc attccqtgct tgcatcatct aggacatqqc tggagacttt atcttccctg ttttttcaca atttcttgga atcccttaca attcctgaga aatgccattc tgacgtgtgg gggcagcccc aaacaagctt cctcat cact cqgcccaaga cgattccgac gctctgtttg caggeggcat gactgtgttt aattcggctg catgqtgttg WO 01/34780 ggttcgcagg atgggtcttt cacctctctc gataacaagg cacaatgtaa gcaaaagtag tccgtgagac gatcgcttca atqtgccccq ttgagatatg caccctgtgc PCTUSOOI3 1254 agaaggqagg taactggcgg tctccccaca aggcagggg tctcagaagg acgttgtata agacaaacaa atgagagaca gaaatgaatt attgggaatt acagtttgcc cgatttgagg acaggacacg tctattacaa gcctcttaca actacggatg tggaggttat caaagaagag tgagcctttt cgcaaggttg aatggaggcg tttactactt ctgagtgagg acagcctcgg aggcttcgca tggagtqaaa qtagccccca actattccca tattttccta tctttcatcc gaaatggaat gatgaacgca aaacacgttc aggagattgc cattagccac aagagtgtga taaaaagtgt taaatggagg aaggatqgaa gtccagagag ccttcggcca tatttctata ccaacat gta ctcctacatg agacaatctt cattctgaag aaaacttaga qggctattta atttaagaaa ggttcattac atttgatcca gggtaatCgg tcatttggtt cttcattcct a 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1491 <210> <211> 1497 (212> DNA <213> Taxus cuspidata <400> atggaCgcc tccactgaat ctcttccqtt ttcattggcg tttgacgaga acagtagtac g tgca ga t t aggaggggtg gcgctqcaqa tggaagggaa tcggccatct ttggaaacta cat agagcac agaaaagaag tcactttcag tcttctctgc ctcttgtctt tccaacaaag tqqcaagtag gccatcactg acttacagta agattcgatc ca acggt cat tttgtcaaaa ccactccctc tgtataagag ctttttccat ctaaacgcca agtcgtt tat qagtgaagaa tctgcggccc cqtggcccgc aagaccatat gttacattqg aagatgaggt tgtttttcaa ttctggtcgg tccagggacg attgcagtct agatgacaaa tccatgcctc ccaatccaga aggagggcga ctcaggaaac acattcagta cacatcccaa aggaagg.aaa gtgtgggatg cttttagcag ctcCtccttc cacagttgca tgccctctca ctcctccctt cttcctgagg attcggCCc tgcgggaaaC tcaatttatg agttatgcgc taaaatgaat gaatgtactt catatatgat aagttttgct gccaagctc ggatcggcaa gggact ccct ctatgacacc atgctatCaa agaaatcaca gctgcggatg tgatqgtaCC ggaCttgtat gcatgtaqct ggaattttca ctacaczccca caagggattt aaatttaatg gctattgctg aaaCttcctc gctcttcqat gtgttcaaga cggcttattC aagctcatgg tctgctcttg acagagatcc cctttggtaa aagcaggaa c cttccgattg aacaaaatta caqccacgca cacccaatgg accacttcgc aaagtagtt c tggaaggatc tttcctccag aattccaaaa ttcaatgaac ccttacacat aagatggaga gttgatcccg tccattaaac aggtcacaca gtattcttct ctgggaaatt cgaactcgct cctccttqat tgtccaacga gggagaattc caggtttttt agagtcatat gagaqctcgt aggatcgtct acttgcccgg tgctgtcttt ggatctgctc atyagatact caatggcttt aagagcaatt tcaaagCCat ttttcggaac gggggaagct cagagaaatt ttttgccctt tattactatt acgaaaa.Sat tgtttccgag gctggactgt gcttctcCtg aggcatccct ggagcaattt tgggcatccc ggagaagCtg cgttgccaCC cggccctggt caacgaaaaa cttcaacatt gcataagctt atttggtttC aattaaaaag tttgttttgC cgacaacttt gattttcaag ggagat cctt gaaatacaca atttcgcaag gttgtggaca catgccttca cggtggaggc cgttcatcat atcaggggat accatag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 13B0 1440 1497 <210> 86 <211> 1461 <212> DNA <213> Taxus cuspidata <400> 86 atggagcagC tttatctgtg gccaataaac Cqttttctta agqtatqqqc gatcccgagt ct cqcgccct caaaggaagc ttcatggaag taatctatag taattttact ccaa acttccC gagacgctaa cgattttCag tcaataagta tcagaaatct tccatgcaac atatacaaga tat tgtctat gttattaaga acctggatca atcgcctgqa atgtagtttg cgtcttgcaa tatcggcaaa tgctgtcaat catct ttcag tccaattggt cggagtaatg gctggattgc cggcgaaagt tttggaagaa aatgagggaa tatggattgt ttgttgaagc gctggaatga atttatgggt acagacaagg catttattgg tctttgatga cacgtgcagt gctgttcga cqgcggtaca atgagaCgCt gaaaa tggga tttgggactg gaatggatcc agagactatc acatgagctc tgtgtcggtg atccaacgca gggggaactt cagctctgac ggaggaggga WO 01/34780 gacatcccta ctggacttac ggagctgtcc gccaggggaa gaggtgctcc attattgcag atqacqtttg gagcatgacg taccaatcaa accgtggttc aaaggatgqa gaagctgaca ccatgttata tttgaaattg attgatcgtg tat tctcgag PCTUSOO/31 254 ttcaacacaa ctccatcaqa tctcttt ccc ttctgttaaa gcaatgactt atacaataat ctgtaaagta ctcttttgaa.

tgaaattcgt ttttcaggga ccgtttctgt aattcctccc tgccatttgg ctctctttct cgacttactt tacacgaatg gtgcaatcag agaaatggga cctcaatatc aaqaattcac qttgaccaaa cttttttgtg cctcgctgag ggccaaaggg tcattgtgta agccaaacaa tttcttgaqc ttggcgctgg aagaggtggc tcacaactt t tcctcttcct attgttctga catatttata cctggaacca aagtgtataa cttgtgaggg tttgctggtg aatccacgag aaaggcaatg ataaatgaaa gatattaaag gccacacatg caaaatgagg aggctctgtc gtcactaaat tccacagaaa acttgatggc aagctttcga cttatgcgag aggagaggag agggcacatt tcgaaacttc cactggagga aaaagctgac cacttcgtct t gaa aga tt t ttgatggaaa gtcaagaaac caggactcca tcagatggga atggttttcc gaagagattg cgatttcgtg aggaat tcgg agaacatcca ttcggacgaa agcaatggcc gttgagggct gtggaatgac gggt ggtgca tqttattccc ataccattat.

gttqgaggag tttggcaaga gcagctggaa aatccgtctc 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1461 <210> 87 <211> 512 <212> PRT <213> Taxus cuspidata <400> 87 Met Asp Ala Phe Asn 1 5 Val Leu Met Gly Pro Leo Ala Lys Phe 10 Asp Asn Phe Met Gin Val Thr Ala Gly Ser Tyr Ser Asn Leo Sex Val Thr Ile Thr Ile Arg Ser le Ala Val Ile Leu Leu Leo Val Lys Pro Gin Ser Cys Val Leo Pro Pro Gly Leo Gly Tyr Pro Phe Ile Gly Gbu Thr Pro Gin Gin Phe Phe Gin Leu Leo Gin Phe Arg Ser Asn Asp Gbu Arg Gin Lys Phe Gly Sex Val Phe Lys Thr Ser Gly Asn Arg 115 Ile Gly Asp Arg Val Val Leu Cys Gly Pro Ser 110 Giu Ala Ser Leu Leu Leu Ser Glu Asn Lys Leu Trp Pro 130 Sex Ser Ser Ile Leo le Giy Glu Se Ile Ala Gly Asn Gly Glu Lys Arg Ilie Leo Arg Ala Ala Val Asn Arg 155 Leo Gly Pro Giy Leo Gin Asn Tyr Ala Lys Met Arg Ser Glu 175 Ile Glu His Met Asn Gbu Lys Trp Lys Gly Lys Glu 185 Gin Val Lys 190 Val Leo Pro Leo Val Lys Glu Asn Val Phe Ser Ile Ala Thr Sex Leu WO 01/34780 195 Phe Phe Gly Val Asn Asp PCTUSOOI3 1254 Gly Glu Arg Glu Arg Leu His Asp Leu 220 Val Phe Ser Ile Pro Leu Asp Phe Pro 235 240 Leu Glu Ala Arg Leu Lys Leu Asp Lys 250 255 Arg Arg Arg Ser Asp Leu Arg Ser Gly 265 270 Leu Leu Ser Val Trp Leu Thr Phe Lys 280 285 Thr Asp Lys Glu Ilie Leu Asp Asn Phe 300 Tyr Asp Thr Thr Thr Ser Ala Leu Thr 315 320 Ser Ser Thr Glu Cys Tyr His Lys Val 330 335 Val Ser Asn Lys Lys Glu Gly Glu Glu 345 350 Asp Met Lys Tyr Thr Trp Gin Val Val 360 365 Pro Pro Leu Phe Gly Ser Phe Arg Lys 380 Asp Gly Tyr Thr Ile Pro Lys Gly Trp 395 400 Ser Thr His Gly Ary Glu Glu Tyr Phe 410 415 Pro Ser Arg Phe Glu Glu Glu Gly Arg 425 430 Leu Pro Phe Gly Ala Gly Val Arg Thr 440 445 Lys Thr Gin Ile Leu Leu Phe Leu His 460 Gly Tyr Ilie Pro Leu Asp Pro Asp Glu 475 480 Pro Pro Leu Pro Ala Asn Gly Phe Ala 490 495 Ser Phe Asp Gin Gly Ser Pro Met Glu 505 510 WO 01/34780 WO 0134780PCTIUSOO/3 1254 <210> 88 <211> 485 <212> PRT <213> Taxus cuspidata <400> 88 Met Asp Ala Leu Lys Gin 1 Thr His Gly Gin Ser Arg Ser Gly Pro 145 Arg Pro Gly Val Ser 225 Thr Ser Arg Ala Ser Th r Ile Ile Val1 Met 115 Gin Al a Ile Val Th r 195 Leu His Le u As n Asn 275 Ser Ilie Phe Arg Gly Phe Ser Ser Asp Val Gly Pro Gin Met Leu Gly 125 Thr Arg 140 Ser Ser Glu Ala Ser Arg His Asn 205 Asn Ile 220 Leu Ala Arg Ala Thr Phe Asp Asn 285 Leu Ser Pro Th r Phe Ala Ser 110 Gly Phe Gly Th r Le u 190 Le u Pro Asp Gly Th r 270 Phe WO 01/34780 Len Leu His Gly Ser Tyr 290 PCTUSOO/3 1254 Ser Thr Asn Ser Pro Leu Thr Met Leu 300 Ala Ser 310 Ile Leu 325 Lys Gin Tyr Pro Tyr Asn Tyr Ser 390 Lys Pro 405 Tyr Leu Ala Lys Ser Gly Len Pro 470 Se r 485 Lys Gi u Val1 Arg 365 Gi y Tyr Gly Arg Leu 445 Asn Ala Ile 335 Gin Ala Lys Lys His 415 Cys His Lys Gin 320 Al a Gin Ile Len Asp 400 Val1 Pro Phe Le u Lys 480 Pro Len Pro Val Asn Gly Leu Pro Ile 475 '210> 89 (211> 493 <212> PRT <213> Taxus <400> 89 Met Asp Ser 1 Val Ile Gin Len Phe Phe Gly Asni Leu Ser Len Arg cuspidata Phe Thr Phe Val Thr Ile Val Giu Tyr Ilie Len Ser 25 Phe Arg Tyr Arg Asn Lys 40 Gly Phe Pro Phe Ilie Gly 55 Ser Gin Thr Pro Gin Phe 70 Lys Met Gly Lys Ile Trp Gin 10 Len Thr Leu Thr Ala Ile Leu Ser Ser His Lys Leu Pro Pro Gin Thr Ilie Gin Phe Leu Arg Phe Phe Asp Gin Arg Val Lys 75 WO 01/34780 Lys Phe G] Ile Phe CN Lys Leu Vo 12I Glu Asn SE 130 Ser Ala LE 145 Ala Lys Mc Gly Lys GI Ser Ile Al Giu Arg LE 210 Val Arg Le 225 Arg Ser L Ser Asp LE Val Leu LE Glu Ile LE 290 Thr Ile Se 305 Glu Cys T Lys Lys G1 Tyr Thr Tr Phe Gly SE 370 Giy Leu Lys His 140 Leu Asn Arg Asp Ala 220 Lys Ile Gin Pro Al a 300 Leu GI y Leu Met Asn 380 Pro Asn 110 Leu Ile Thr Lys Leu 190 Pro Ser Val Ser Leu 270 Arg Tyr Se r Leu Ala 350 Pro Asp PCT/USOO/3 1254 ,ir Val lu Asp -t Gly ?u Arg rr Ile 160 :p Lys 1 Phe Ln Gin t Ala LU Ala 240 ,g Arg wu Ser -p Giu ip Thr ;n Pro 320 .a Asn !t Lys o Leu y Tyr Thr Ile Pro Lys Gly 385 Ile Val Leu Trp Thr Thr Tyr Ser Thr His WO 01/34780 Val Lys Glu Phe Glu His Gly Gly Gly 435 Ilie Leu Leu 450 Pro Val Asp 465 Pro Ala Asn PCT/USOO/3 1254 Arg Pro Ser Arg 415 Phe Leu Pro Phe 430 Ser Lys Thr Glu 445 Gly Ser Tyr Leu Phe Pro Pro Leu 480 Ser <210> <211> 496 <212> PRT <213> Taxus cuspidata <400> Met Glu Leu Trp Asn Met Phe Leu Pro Trp Ile Ser Ile Ala Thr Ala 1 Ci WO 01/34780 Val Lys Asp 195 Lys Pro Gly 210 Ser Ala Gly 225 Gly Lys Ala Gin Ile Arg Gly Asn Phe 275 Leu Arg Leu 290 Thr Gly Gly 305 His Leu Ser Giu Lys Leu Glu Ile Lys 355 Arg Met Val 370 Val Val Tyr 385 Ser Val Arg Arg Phe Asp Ile Pro Phe 435 Arq Leu Glu 450 Trp Glu Leu 465 His Pro Val Ala Gi u Leu Arg 245 His Asp Glu Asp Se r 325 Asp Val1 Pro Gly Thr 405 Asp Gin Gi u Giu Se r 485 Ala A rg Asp Met 250 His Se r Asp Ala Gin 330 Gi y Asn Phe Lys Giu 410 Arg Cys His Thr Leu 490 Asp Arg Le u 235 Val1 Lys Gin As n Leu 315 A rg Gly Val Lys Gly 395 T yr His Pro Leu Asn 475 Lys Phe 205 Phe Trp Gin Gi y Lys 285 Met Thr Arq Leu Se r 365 Ala Lys Pro Pro Asn 445 Leu Tyr ValI PCTUSOO/31 254 Gly Leu Asp Phe Val Phe 240 Phe Ser 255 Glu Gly Gly Asp Leu Leu Leu Lys 320 Glu Cys 335 Trp Ser Gly Leu Val Asp His Tyr 400 Pro Giu 415 5cr Phe Phe Ala Tyr Asp Ile Pro 480 Pro Thr 495 WO 01/34780 <210> 91 <211> 498 <212> PRT <213> Taxus cuspidata <400> 91 Met Asp Ala Leu Tyr Lys PCTUSOO/31254 Thr Val Ser Phe Leu Leu Lys Leu Leu Arg Phe Gly Leu Cys 105 Leu Val 120 Asn Ser Ala Leu Lys Met Lys Asp 185 Ile Ser 200 Arg Leu Pro Ile Ala Lys Asp Cys 265 Cys Ser 280 Thr Ile Se r Glu Phe Leu Leu Gin G lu Gly 160 His Leu Ile Ile Phe 240 Ser Pro Gly Leu 275 Leu Pro His Pro Met Asp Glu Ile Leu WO 01/34780 290 His Ala SE 305 Leu Leu SE Leu Glu I] Asp Leu L Arg Met P1 370 Ile Gin Tj 385 Thr Tyr SiE Phe Met Pi Thr Phe LE 41 Phe Ser L 450 Phe Ser SE 465 PCT/USOO/31254 Leu Val1 I le Gin 365 Al a Leu Glu Val1 Val 445 Phe I le Phe Giu 335 T rp Thr Thr Trp Glu 415 Pro Trp Lys Gly Pro Leu Pro Pro Leu Pro Ser Lys Gly Phe Ser Ile Lys Leu Phe Pro 485 490 495 Arg Pro <210> 92 <211> 486 <212> PR? <213> Taxus <400> 92 Met Giu Gin 1 Val Leu Gly Asn Asp Arg Gly Ser Ala Asp Ala Lys cuspidata Leu Ile Tyr Ser Ile Vai 5 Leu Phe Ile Cys Vai Ile 25 Gin Gly Asn Gly Ser Ala 40 Gly Leu Pro Phe Ile Gly 55 Ser Pro Gly Arg Arg Lys Tyr Ser Asn Trp Tyr Leu Trp 10 Leu Leu Leu Leu Arg Arg Ser Asn Lys Pro Lys Leu Pro Pro Giu Thr le Arg Phe Leu Arg Phe Phe Asp Glu His Glu Leu WO 01/34780 WO 0134780PCT[USOO/3 1254 Tyr Gly Pro Ile Phe Arg Cys Ser Leu Phe Gly Arg*Thr Arg Ala Val Ser Val Phe Leu Ser Ala Thr His 385 390 WO 01/34780 Glu Ala A! Thr Leu G.

Cys Pro

G:'

Asn Phe V 450 Thr Tyr PI 465 Tyr Ser A: PCTIUSOO/31254 Pro Trp Arq Trp Gin Asn Glu Gly Gin Glu 410 415 Tyr Met Pro Phe Gly Arg Gly Gly Arg Leu.

425 430 Ala Arq Phe Glu Ilie Ala Leu Phe Leu His 440 445 Arg Trp Glu Gin Leu Glu Ile Asp Arg Ala 455 460 Ser Thr Glu Asn Gly Phe Pro Ile Arg Leu 475 480 <210> 93 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer <400> 93 atggcccrta agcaattgga agtttc <210> 94 <~211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 2CR Primer <400> 94 ttaagatctg gaatagagtt taatgg

Claims (6)

1. A purified protein, comprising an amino acid sequence selected from the group consisting of: SEQ ID NOS: 22-42, 56-68, and 87-92.

2. An isolated nucleic acid molecule encoding a protein of claim 1.

3. An isolated nucleic acid molecule of claim 2, further comprising a sequence selected from the group consisting of: SEQ ID NOS:1-21, 81-86, and 43-55.

4. A method of screening for an agent which specifically binds a protein of claim 1, the method comprising the step of contacting the agent with the protein under conditions effective to allow binding of the agent and protein. 15 5. An isolated nucleic acid molecule that: S* hybridizes under high-stringency conditions with a nucleic acid probe, the probe comprising a sequence selected from the group consisting of SEQ ID NOS: 1-21,

43-55, and 81-86 and fragments thereof; and encodes a protein having oxygenase activity. o 6. An isolated nucleic acid molecule that: i has at least 60% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-21, 43-55, and 81-86; and encodes a protein having oxygenase activity. 7. An oxygenase encoded by a nucleic acid molecule of claim 5 or claim 6. 8. A purified protein having oxygenase activity, comprising an amino acid sequence selected from the group consisting of: an amino acid sequence selected from the group consisting of SEQ ID NOS:

56-68 and 87-92; an amino acid sequence that differs from the amino acid sequence specified in by one or more conservative amino acid substitutions; and an amino acid sequence having at least 70% sequence identity to the sequences specified in or B. \rebeccae\keep\apcificatioa\l14887-01 amndmants.doc 24/01/os COMS ID No: SBMI-01090146 Received by IP Australia: Time 16:00 Date 2005-01-24 14/02 2005 13:21 FAX 61 3 91438333 GRIFFITH HACK 0]004/006 66 9. An isolated nucleic acid molecule encoding a protein of claim 8. An isolated nucleic acid molecule of claim 9, further comprising a sequence selected from the group corsisting of SEQ ID NOS: 43-55 and 81-86. 11. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to a nucleic acid sequence of any one of claims 2, 3, 5, 6, 9 or 12. A cell transformed with a recombinant nucleic acid molecule of claim 11. 13. A transgenic non-human organism, comprising a recombinant nucleic acid molecule of clain 11, wherein the transgenic organism is selected from the group consisting of plants, bacteria, insects, fungi, and mammals. 14. A method for isolating a nucleic acid sequence, comprising: hybridizing under high-stringency conditions the nucleic acid sequence to at *6 •least 10 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOS: 1-21, 43-55, and 81-86; and 20 identifying the nucleic acid sequence as one that encodes an oxygenase. S 15. A method of claim 14, wherein hybridizing the nucleic acid sequence is performed under high-stringency conditions. 16. A method of claim 14 or claim 15, wherein step occurs in a PCR reaction. 17. A method of claim 14 or claim 15, wherein step occurs during library screening. 18. A method of any one of claims 14 to 17, wherein the isolated nucleic acid sequence is isolated from the genus Taxus. 19. A nucleic acid sequence isolated by a method of any one of claims 14 to 18. 20. A purified oxygenase encoded by a nucleic acid sequence of claim 19. 21. A method of screening for an agent which specifically binds an oxygenase of 34.rccht\.Egqp\4mun,,o1 dW.plJ,ntia.daC 14 /0 COMS ID No: SBMI-01118913 Received by IP Australia: Time 13:23 Date 2005-02-14 24/01 2005 15:53 FAX 61 3 92438333 GRIFFITH HACK SIPAUSTRALIA lih 01 67 claim 7 or claim 20, the method comprising the step of contacting the agent with the oxygenase under conditions effective to allow binding of the agent and oxygenase 22. A method for synthesizing a second intermediate in the Taxol biosyntheticpathway, comprising: contacting a first intermediate with an oxygenase of claim 7 or claim 20; and allowing the oxygenase to transfer at least one oxygen atom group to the first intermediate, wherein transfer of the at least one oxygen atom group yields the second intermediate in the Taxol biosynthetic pathway. 23. A method of claim 22, wherein the oxygenase is produced by an introduced oxygenase gene in a transgenic organism, and step occurs in vivo. 24. A method for transferring an oxygen atom to a taxoid, comprising: 1 5 contacting a taxoid with at least one oxygenase of claim 7 or claim 20; and allowing the oxygenase to transfer an oxygen atom to the taxoid. 25. A method of claim 24, wherein the oxygenase is produced by an introduced oxygenase gene in a transgenic organism, and synthesis of the taxoid occurs in vivo. 26. A method of claim 24 or claim 25, wherein at least one paclitaxel molecule is .i produced. 4*55 27. A method of any one of claims 24 to 26, wherein the taxoid is an acylation or a 25 glycosylation variant of paclitaxel. 28. A method of claim 27, wherein the variant of paclitaxel is selected from the group Si consisting of cephalomannine, xylosyl paclitaxel, 10-dactyl paclitaxel, or paclitaxel C. 29. A method of any one of claims 24 to 26, wherein the taxoid is baccatin UI. A method of any one of claim 24 to 26 or 29, wherein the taxoid is an acylation or a glycosylation variant of bassatin II. 31. A method of claim 30, wherein the variant of baccatin III is selected from the group consisting of 7-xylosyl baccatin III or 2-debenzoyl baccatin III. Bt\rebeccaa\k.ep\apecifioaio \14887-01 arndments .doc 24/01/05 COMS ID No: SBMI-01090146 Received by IP Australia: Time 16:00 Date 2005-01-24 24/01 2005 15:54 FAX 61 3 92438333 GIFT AKIASRLAIJ1 GRIFFITH HACK 4 IPAUSTRALIA Z 014 68 32. A method of any one of claims 24 to 26, wherein the taxoid islO-deacetyl- baccatin 111. 33. A method of any one of claims 24 to 26 or 32, wherein the taxoid is an acylation or a glycosylation variant of lO-deacetyl-baccatin I11. 34. A method of claim 33, wherein the variant of baccatin MI is selected from the group consisting of 7-xylosyl 1 0-baccatin III or 2-debenzoyl I 0-baccatin III A protein of claim I or claim 8, substantially as herein described with reference to any of the examples or figures. 36. A nucleic acid molecule of claim 5 or claim 6, substantially as herein described 15 with reference to any of the examples or figures. 37. A method of claim 14, substantially as herein described with reference to any of the examples or figures. 20 Dated this 24th day of January 2005 WASHINGTON STATE UNIVERSITY RESEARCH FOUNDATION By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia B.\rebeoca.\kep\pcifictien \1487-01 nd~ntv-doc 24/01/05 COMS ID No: SBMI-01090146 Received by IP Australia: Time 16:00 Date 2005-01-24