# Patent

## Title
Transacylases of the paclitaxel biosynthetic pathway

## Abstract

Transacylase enzymes and the use of such enzymes to produce paclitaxel, related taxoids, as well as intermediates in the paclitaxel biosynthetic pathway are disclosed. Also disclosed are nucleic acid sequences encoding the transacylase enzymes.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is the U.S. National Stage of International Application No. PCT/US00/27006, filed Sep. 29, 2000 published as PCT Publication No. WO 01/23586, which is a continuation-in-part of U.S. application Ser. No. 09/457,046, filed Dec. 7, 1999, now issued as U.S. Pat. No. 6,287,835, and a continuation-in-part of U.S. application Ser. No. 09/411,145, filed Sep. 30, 1999, now abandoned. Each of the foregoing applications are incorporated in its entirety.


ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with government support under National Cancer Institute Grant No. CA-55254. The government has certain rights in this invention.


FIELD OF THE INVENTION
The invention relates to transacylase enzymes and methods of using such enzymes to produce paclitaxel and related taxoids.
INTRODUCTION
The complex diterpenoid 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, TaxolTM: Science and Applications, CRC Press, Boca Raton, 397–415, 1995; Holmes et al., ACS Symposium Series 583:31–57, 1995). Paclitaxel was originally isolated from the bark of the Pacific yew (Taxus brevifolia). For a number of years, paclitaxel 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 paclitaxel production to be developed. Paclitaxel is currently produced primarily by semisynthesis from advanced taxane metabolites (Holton et al., TaxolTM: 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 paclitaxel 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 (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 paclitaxel 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 paclitaxel biosynthetic pathway is considered to involve more than 12 distinct steps (Floss and Mocek, TaxolTM: 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 paclitaxel 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. 1). The cyclized intermediate subsequently undergoes modification involving at least eight oxygenation steps, a dehydrogenation, an epoxide rearrangement to an oxetane, and several acylations (Floss and Mocek, TaxolTM: Science and Applications, CRC Press, Boca Raton, 191–208, 1995; 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 corresponding cDNA clone isolated and expressed (Wildung and Croteau, J. Biol. Chem. 271:9201–9204, 1996).
The second specific step of paclitaxel biosynthesis is an oxygenation reaction catalyzed by taxadiene-5α-hydroxylase (FIG. 1). The enzyme, characterized as a cytochrome P450, has been demonstrated in Taxus microsome preparations to catalyze the stereospecific hydroxylation of taxa-4(5),11(12)-diene, with double bond rearrangement, to taxa-4(20),11(12)-dien-5α-ol (Hefner et al., Chem. Biol. 3:479–489, 1996).
The third specific step of paclitaxel biosynthesis appears to be the acetylation of taxa-4(20),11(12)-dien-5α-ol to taxa-4(20),11(12)-dien-5α-yl acetate by an acetyl CoA-dependent transacetylase (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999), since the resulting acetate ester is then further efficiently oxygenated to a series of advanced polyhydroxylated paclitaxel metabolites in microsomal preparations that have been optimized for cytochrome P450 reactions (FIG. 1). The enzyme has been isolated from induced yew cell cultures (Taxus canadensis and Taxus cuspidata), and the operationally soluble enzyme was partially purified by a combination of anion exchange, hydrophobic interaction, and affinity chromatography on immobilized coenzyme A resin. This acetyl transacylase has a pI and pH optimum of 4.7 and 9.0, respectively, and a molecular weight of about 50,000 as determined by gel-permeation chromatography. The enzyme shows high selectivity and high affinity for both cosubstrates with Km values of 4.2 μM and 5.5 μM for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced paclitaxel precursors, 10-deacetylbaccatin III or baccatin III. This acetyl transacylase is insensitive to monovalent and divalent metal ions, is only weakly inhibited by thiol-directed reagents and Co-enzyme A, and in general displays properties similar to those of other O-acetyl transacylases. This acetyl CoA:taxadien-5α-ol O-acetyl transacylase from Taxus (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999) appears to be substantially different in size, substrate selectivity, and kinetics from an acetyl CoA:10-hydroxytaxane O-acetyl transacylase recently isolated and described from Taxus chinensis (Menhard and Zenk, Phytochemistry 50:763–774, 1999).
Acquisition of the gene encoding the acetyl CoA:taxa-4(20),11(12)-dien-5α-ol O-acetyl transacylase that catalyzes the first acylation step of paclitaxel biosynthesis and genes encoding other acyl transfer steps would represent an important advance in efforts to increase paclitaxel yields by genetic engineering and in vitro synthesis.
SUMMARY OF THE INVENTION
The invention stems from the discovery of twelve amplicons (regions of DNA amplified by a pair of primers using the polymerase chain reaction (PCR)). These amplicons can be used to identify transacylases, for example, the transacylases shown in SEQ ID NOs: 26, 28, 45, 50, 52, 54, 56, and 58 that are encoded by the nucleic acid sequences shown in SEQ ID NOs: 25, 27, 44, 49, 51, 53, 55, and 57. These sequences are isolated from the Taxus genus, and the respective transacylases are useful for the synthetic production of paclitaxel and related taxoids, as well as intermediates within the paclitaxel biosynthetic pathway. The sequences can be also used for the creation of transgenic organisms that either produce the transacylases for subsequent in vitro use, or produce the transacylases in vivo so as to alter the level of paclitaxel and taxoid production within the transgenic organism.
Another aspect of the invention provides the nucleic acid sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 and the corresponding amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, respectively, as well as fragments of the nucleic acid and the amino acid sequences. These sequences are useful for isolating the nucleic acid and amino acid sequences corresponding to full-length transacylases. These amino acid sequences and nucleic acid sequences are also useful for creating specific binding agents that recognize the corresponding transacylases.
Accordingly, another aspect of the invention provides for the identification of transacylases and fragments of transacylases that have amino acid and nucleic acid sequences that vary from the disclosed sequences. For example, the invention provides transacylase amino acid sequences that vary by one or more conservative amino acid substitutions, or that share at least 50% sequence identity with the amino acid sequences provided while maintaining transacylase activity.
The nucleic acid sequences encoding the transacylases and fragments of the transacylases can be cloned, using standard molecular biology techniques, into vectors. These vectors can then be used to transform host cells. Thus, a host cell can be modified to express either increased levels of transacylase or decreased levels of transacylase.
Another aspect of the invention provides methods for isolating nucleic acid sequences encoding full-length transacylases. The methods involve hybridizing at least ten contiguous nucleotides of any of the nucleic acid sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 44, 49, 51, 53, 55, and 57 to a second nucleic acid sequence, wherein the second nucleic acid sequence encodes a transacylase. 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 transacylases identified by this method.
Yet another aspect of the invention involves methods of adding at least one acyl group to at least one taxoid. These methods can be practiced in vivo or in vitro, and can be used to add acyl groups to various intermediates in the paclitaxel biosynthetic pathway, and to add acyl groups to related taxoids that are not necessarily in a paclitaxel biosynthetic pathway.
SEQUENCE LISTINGS
The nucleic 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 NO: 1 is the nucleotide sequence of Probe 1.
SEQ ID NO: 2 is the deduced amino acid sequence of Probe 1.
SEQ ID NO: 3 is the nucleotide sequence of Probe 2.
SEQ ID NO: 4 is the deduced amino acid sequence of Probe 2.
SEQ ID NO: 5 is the nucleotide sequence of Probe 3.
SEQ ID NO: 6 is the deduced amino acid sequence of Probe 3.
SEQ ID NO: 7 is the nucleotide sequence of Probe 4.
SEQ ID NO: 8 is the deduced amino acid sequence of Probe 4.
SEQ ID NO: 9 is the nucleotide sequence of Probe 5.
SEQ ID NO: 10 is the deduced amino acid sequence of Probe 5.
SEQ ID NO: 11 is the nucleotide sequence of Probe 6.
SEQ ID NO: 12 is the deduced amino acid sequence of Probe 6.
SEQ ID NO: 13 is the nucleotide sequence of Probe 7.
SEQ ID NO: 14 is the deduced amino acid sequence of Probe 7.
SEQ ID NO: 15 is the nucleotide sequence of Probe 8.
SEQ ID NO: 16 is the deduced amino acid sequence of Probe 8.
SEQ ID NO: 17 is the nucleotide sequence of Probe 9.
SEQ ID NO: 18 is the deduced amino acid sequence of Probe 9.
SEQ ID NO: 19 is the nucleotide sequence of Probe 10.
SEQ ID NO: 20 is the deduced amino acid sequence of Probe 10.
SEQ ID NO: 21 is the nucleotide sequence of Probe 11.
SEQ ID NO: 22 is the deduced amino acid sequence of Probe 11.
SEQ ID NO: 23 is the nucleotide sequence of Probe 12.
SEQ ID NO: 24 is the deduced amino acid sequence of Probe 12.
SEQ ID NO: 25 is the nucleotide sequence of the full-length acyltransacylase clone TAX2 (also referred to herein as “TAX02”).
SEQ ID NO: 26 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX2.
SEQ ID NO: 27 is the nucleotide sequence of the full-length acyltransacylase clone TAX1 (also referred to herein as “TAX01”).
SEQ ID NO: 28 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX1.
SEQ ID NO: 29 is the amino acid sequence of a transacylase peptide fragment.
SEQ ID NO: 30 is the amino acid sequence of a transacylase peptide fragment.
SEQ ID NO: 31 is the amino acid sequence of a transacylase peptide fragment.
SEQ ID NO: 32 is the amino acid sequence of a transacylase peptide fragment.
SEQ ID NO: 33 is the amino acid sequence of a transacylase peptide fragment.
SEQ ID NO: 34 is the AT-FOR1 PCR primer.
SEQ ID NO: 35 is the AT-FOR2 PCR primer.
SEQ ID NO: 36 is the AT-FOR3 PCR primer.
SEQ ID NO: 37 is the AT-FOR4 PCR primer.
SEQ ID NO: 38 is the AT-REV1 PCR primer.
SEQ ID NO: 39 is an amino acid sequence variant that allowed for the design of the AT-FOR3 PCR primer.
SEQ ID NO: 40 is an amino acid sequence variant that allowed for the design of the AT-FOR4 PCR primer.
SEQ ID NO: 41 is a consensus amino acid sequence that allowed for the design of the AT-REV1 PCR primer.
SEQ ID NO: 42 is a PCR primer, useful for identifying transacylases.
SEQ ID NO: 43 is a PCR primer, useful for identifying transacylases.
SEQ ID NO: 44 is the nucleotide sequence of the full-length acyltransacylase clone TAX6 (also referred to herein as “TAX06”).
SEQ ID NO: 45 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX6.
SEQ ID NO: 46 is a PCR primer, useful for identifying TAX6.
SEQ ID NO: 47 is a PCR primer, useful for identifying TAX6.
SEQ ID NO: 48 is a 6-amino acid motif commonly found in transacylases.
SEQ ID NO: 49 is the nucleotide sequence of the full-length acyltransacylase clone TAX5 (also referred to herein as “TAX05”).
SEQ ID NO: 50 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX5.
SEQ ID NO: 51 is the nucleotide sequence of the full-length acyltransacylase clone TAX7 (also referred to herein as “TAX07”).
SEQ ID NO: 52 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX7.
SEQ ID NO: 53 is the nucleotide sequence of the full-length acyltransacylase clone TAX10.
SEQ ID NO: 54 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX10.
SEQ ID NO: 55 is the nucleotide sequence of the full-length acyltransacylase clone TAX12.
SEQ ID NO: 56 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX 12.
SEQ ID NO: 57 is the nucleotide sequence of the full-length acyltransacylase clone TAX13.
SEQ ID NO: 58 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX13.
SEQ ID NO: 59 is the nucleotide sequence of the full-length acyltransacylase clone TAX9 (also referred to herein as “TAX09”).
SEQ ID NO: 60 is the deduced amino acid sequence of the full-length acyltransacylase clone TAX9.
SEQ ID NO: 61–76 are the amino acid sequences shown in FIG. 6.



FIGURES
FIG. 1 shows an outline of the paclitaxel biosynthetic pathway. The cyclization of geranylgeranyl diphosphate to taxadiene by taxadiene synthase and the hydroxylation to taxadien-5α-ol by taxadiene 5α-hydroxylase (a), the acetylation of taxadien-5α-ol by taxa-4(20),11(12)-dien-5α-ol-O-acetyl transferase (gene product of TAX1) (b), the conversion of a 2-debenzoyl “taxoid-type” intermediate to 10-deacetylbaccatin III by a taxane-2α-O-benzoyl transferase (gene product of TAX2; SEQ ID NO: 25) (c), the conversion of 10-deacetylbaccatin III to baccatin III by 10-deacetylbaccatin III-10-O-acetyl transferase (gene product of TAX6) (d), and the side chain attachment to baccatin III to form paclitaxel (e) are illustrated. The broken arrow indicates several as yet undefined steps.
FIG. 2 shows the peptide sequences generated by endolysc and trypsin proteolysis of purified taxadienol acetyl transacylase.
FIGS. 3A–3D include elution profiles from several different columns. FIG. 3A is an elution profile of the acetyl transacylase on Source HR 15Q (10×100 mm) preparative scale anion-exchange chromatography; FIG. 3B is an elution profile on analytical scale Source HR 15Q (5×50 mm) column chromatography; and FIG. 3C is an elution profile on the ceramic hydroxyapatite column. The solid line is the UV absorbance at 280 nm; the dotted line is the relative transacetylase activity (dpm); and the hatched line is the elution gradient (sodium chloride or sodium phosphate). FIG. 3D is a photograph of a silver-stained 12% SDS-PAGE showing the purity of taxadien-5α-ol acetyl transacylase (50 kDa) after hydroxyapatite chromatography. A minor contaminant is present at  ̃35 kDa.
FIG. 4 shows four forward (AT-FOR1, AT-FOR2, AT-FOR3, AT-FOR4) and one reverse (AT-REV 1) degenerate primers that were used to amplify an induced Taxus cell library cDNA from which twelve hybridization probes were obtained. Inosine positions are indicated by “I”. Each of the forward primers was paired with the reverse primer in separate PCR reactions. Primers AT-FOR1 (SEQ ID NO: 34) and AT-FOR2 (SEQ ID NO: 35) were designed from the tryptic fragment SEQ ID NO: 30; the remaining primers were derived by database searching based on SEQ ID NO: 30.
FIGS. 5A–5G shows data obtained from a coupled gas chromatographic-mass spectrometric (GC-MS) analysis of the biosynthetic taxadien-5α-yl acetate formed during the incubation of taxadien-5α-ol with soluble enzyme extracts from isopropyl β-D-thiogalactoside (IPTG)-induced E. coli JM109 cells transformed with full-length acyltransacylase clones TAX1 (SEQ ID NO: 28) and TAX2 (SEQ ID NO: 25). FIGS. 5A and 5B show the respective GC and MS profiles of authentic taxadien-5α-ol; FIGS. 5C and 5D show the respective GC and MS profiles of authentic taxadien-5α-yl acetate; FIG. 5E shows the GC profile of taxadien-5α-ol (11.16 minutes), taxadien-5α-yl acetate (11.82 minutes), dehydrated taxadien-5α-ol (“TOH—H2O” peak), and a contaminant, bis-(2-ethylhexyl)phthlate (“BEHP” peak, a plasticizer, CAS 117-81-7, extracted from buffer) after incubation of taxadien-5α-ol and acetyl coenzyme A with the soluble enzyme fraction derived from E. coli JM109 transformed with the full-length clone TAX1 (SEQ ID NO: 27). Panel F shows the mass spectrum of biosynthetically formed taxadien-5α-yl acetate by the recombinant enzyme (11.82 minute peak in GC profile FIG. 5E); FIG. 5G shows the GC profile of the products generated from taxadien-5α-ol and acetyl coenzyme A by incubation with the soluble enzyme fraction derived from E. coli JM 109 cells transformed with the full-length clone TAX2 (note the absence of taxadien-5α-yl acetate indicating that this clone is inactive in the transacylase reaction).
FIGS. 6A–6N show a pileup of deduced amino acid sequences listed in Table 1, and of TAX1 (SEQ ID NO: 28) and TAX2 (SEQ ID NO: 25). Residues underlined in bold italics (and bold italics) indicate the few regions of conservation. Forward arrow (left to right) shows conserved region from which degenerate forward PCR primers were designed. Reverse arrow (right to left) shows region from which the reverse PCR primer was designed (cf., FIG. 4).
FIG. 7 shows a dendrogram of the deduced peptide sequence relationships between Taxus transacylase sequences (Probes 1–12, TAX1 (SEQ ID NO: 28), and TAX2) and closest relative sequences of defined and unknown function obtained from the GENBANKTM database described in Table 1.
FIG. 8 shows a postulated biosynthetic scheme for the formation of the oxetane, present in paclitaxel and related late-stage taxoids, in which the 4(20)-ene-5α-ol is converted to the 4(20)-ene-5α-yl acetate followed by epoxidation to the 4(20)-epoxy-5α-acetoxy group and then intramolecular rearrangement to the 4-acetoxy oxetane moiety.
FIGS. 9A and 9B show radio-HPLC (high-performance liquid chromatography) analysis of the biosynthetic product (Rt=7.0±0.1 minutes) generated from 10-deacetylbaccatin III and [2-3H]acetyl CoA by the recombinant acetyl transferase (gen product of TAX6 (SEQ ID NO: 45)). FIG. 9A shows the UV profile and FIG. 9B shows the coincident radioactivity profile, both of which coincide with the retention time of authentic baccatin III. For the enzyme preparation, E. coli cells transformed with the pCWori+ vector harboring the putative DBAT gene were grown overnight at 37° C. in 5 mL Luria-Bertani medium supplemented with ampicillin, and 1 mL of this inoculum was added to and grown in 100 mL Terrific Broth culture medium (6 g bacto-tryptone, Difco Laboratories, Spark, Md., 12 g yeast extract, EM Science, Cherryhill, N.J., and 2 mL gycerol in 500 mL water) supplemented with 1 mM IPTG, 1 mM thiamine HCl and 50 μg ampicillin/mL. After 24 hours, the bacteria were harvested by centrifugation, resuspended in 20 mL of assay buffer (25 mM Mopso, pH 7.4) and then disrupted by sonication at 0–4° C. The resulting homogenate was centrifuged at 15,000 g to remove debris, and a 1 mL aliquot of the supernatant was incubated with 10-deacetylbaccatin III (400 μM) and [2-3H]acetyl coenzyme A (0.45μCi, 400 μM) for 1 hour at 31° C. The reaction mixture was extracted with ether and the solvent concentrated in vacuo. The crude product (pooled from five such assays) was purified by silica gel thin-layer chromatography (TLC; 70:30 ethyl acetate:hexane). The band co-migrating with authentic baccatin III (Rf=0.45 for the standard) was isolated and analyzed by radio-HPLC to reveal the new radioactive product described above. Extracts of E. coli transformed with empty vector controls did not yield detectable product when assayed by identical methods.
FIGS. 10A and 10B show the results from a combined reverse-phase HPLC-atmospheric pressue chemical ionization MS (mass spectrometry) analysis of (FIG. 10A) the biosynthetic product (Rt=8.6±0.1 minutes) generated by recombinant acetyl transferase with 10-deaceylbaccatin III and acetyl CoA as co-substrates, and of (FIG. 10B) authentic baccatin III (Rt=8.6±0.1 minutes). The diagnostic mass spectral fragments are at m/z 605 (M+NH4+), 587 (MH+), 572 (MH+—CH3), 527 (MH+—CH3COOH), and 509 (MH+—(CH3COOH+H2O)). For preparation of recombinant enzyme and product isolation, see FIGS. 9A and 9B legend.
FIG. 11 shows an outline of the synthesis and utilization of 2-debenzoyl-7,13-diacetylbaccatin III: methylene chloride, acetic anhydride, 4-(N,N′-dimethylamino)pyridine, triethylamine, 25° C., 18 h (a), tetrahydrofuran, Red-Al (65+% in toluene), 0° C., 30 minutes (b), and the reaction catalyzed by taxane-2α-O-benzoyl transferase in the presence of benzoyl coenzyme A (c).
FIGS. 12A and 12B show radio-HPLC analysis of the biosynthetic product (Rt=21.9 ±0.1 minute) generated from 2-debenzoyl-7,13-diacetylbaccatin III and [7-14C]benzoyl CoA by the recombinant TAX2 benzoyl transferase (SEQ ID NO: 25). FIG. 12A shows the coincident UV profile (A228) and FIG. 12B shows the radioactivity profile (in mV), both of which coincide exactly with the retention time of authentic 7,13-diacetylbaccatin III. Extracts of control E. coli transformed with empty vector did not yield detectable product when assayed by identical methods.
FIGS. 13A and 13B show the results from a coupled reverse-phase HPLC-atmospheric pressure chemical ionization MS analysis of (FIG. 13A) the biosynthetic product (Rt=43.6±0.1 minute) generated by the TAX2 (SEQ ID NO: 25) recombinant benzoyl transferase with 2-debenzoyl-7,13-diaceylbaccatin III and benzoyl CoA as co-substrates, and of (FIG. 13B) authentic 7,13-diacetylbaccatin III (Rt=43.6±0.1 minute). The diagnostic ions are at m/z 688 (P+NH4+), 671 (PH+), 652 (P+—H2O), 611 (PH+—CH3COOH), 593 (m/z 652-CH3COO), 551 (m/z 611-CH3COOH), and 489 (m/z 611-PhCOOH).
FIG. 14 shows a dendrogram of the pairwise relationship among the deduced amino acid sequences of isolated, full-length T. cuspidata acyl transferases designated TAX01, TAX02, TAX05, TAX06, TAX07, TAX09, TAX10, TAX12, TAX 13, (SEQ ID NOS: 28, 26, 50, 45, 52, 60, 54, 56, and 58, respectively) and of PCHCBT (designating the anthranilate hydroxycinnamoyl/benzoyl transferase of Dianthus (protein identification number [PID] cab06427 and GENBANKTM accession no. Z84383 located in the ENTREZTM databank)). Distance along the vertical axis is proportional to the difference between sequences; distance along the horizontal axis has no significance.
FIGS. 15A–15D show the deduced amino acid sequence comparison of six transacylases (TAX05, TAX07, TAX09, TAX10, TAX12, and TAX13) as well as TAX01 (taxadien-5α-ol acetyl transferase, accession no. AF190130), TAX02 (taxane-2α-O-benzoyl transferase, accession no. AF297618), TAX06 (10-deacetylbaccatin III-0-O-acetyl transferase, accession no. AF193765) and PCHCBT (anthranilate hydroxycinnamoyl/benzoyl transferase, accession no. Z84383). Residues underlined in bold italics indicate positional identity for at least three of the compared sequences; similar amino acids are indicated by bold italics. The alignment was created with the PileUp program (Wisconsin Package Version 10; Genetics Computer Group, Madison, Wis.).



DETAILED DESCRIPTION
I. Definitions
Mammal: This term includes both humans and non-human mammals. Similarly, the term “patient” includes both humans and veterinary subjects.
Taxoid: A “taxoid” is a chemical based on the Taxane ring structure as described in Kinston 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, i.e., 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.
Orthologs: An “ortholog” is a gene that encodes a protein that displays a function that is similar to a gene derived from a different species.
Homologs: “Homologs” are two nucleotide sequences that share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into 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 single- or double-stranded, and that may be based on a complete or partial naturally occurring nucleotide sequence encoding one or more of the transacylase genes of the present invention. It is understood that such nucleotide sequences include intentionally manipulated nucleotide sequences, e.g., 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 transacylase encoded by the nucleotide sequence maintains transacylase 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 often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. “Recombinant” is also 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 transacylases 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.
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 be prepared readily 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. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed in, e.g., Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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 10 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 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, e.g., 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. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al. (eds.), 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, Mass.). 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 having a higher specificity than a corresponding primer of only 15 nucleotides. 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., Nuc. Acids Res. 16:10881–10890, 1988; Huang, et al., CABIOS 8:155–165, 1992; and Pearson et al., Meth. in Mol. Biol. 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 (BLASTTM, 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 Internet, for use in connection with the sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx. BLASTTM can be accessed on the internet. A description of how to determine sequence identity using this program is available on the internet.
For comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function of the BLASTTM program is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). 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.
Transacylase (an older name for acyltransferase) activity: Enzymes exhibiting transacylase activity are capable of transferring acyl groups, forming either esters or amides, by catalyzing reactions in which an acyl group that is linked to a carrier (acyl-carrier) is transferred to a reactant, thus forming an acyl group linked to the reactant (acyl-reactant).
Transacylases: Transacylases are enzymes that display transacylase activity as described supra. However, all transacylases do not recognize the same carriers and reactants. Therefore, transacylase enzyme-activity assays must utilize different substrates and reactants depending on the specificity of the particular transacylase enzyme. One of ordinary skill in the art will appreciate that the assay described below is a representative example of a transacylase activity assay, and that similar assays can be used to test transacylase activity directed towards different substrates and reactants.
Substantial similarity: A first nucleic acid is “substantially similar” to a second nucleic acid if, when optimally aligned (with appropriate nucleotide deletions or gap insertions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about, for example, 50%, 75%, 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 BLASTTM sequence analysis software (blastn) available from The National Center for Biotechnology Information. Such comparisons may be made using the software set to default settings (expect=10, filter=default, descriptions=500 pairwise, alignments=500, alignment view=standard, gap existence cost=11, per residue existence=1, per residue gap cost=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 BLASTTM software (blastp) using default settings.
II. Characterization of acetyl CoA:taxa-4(20),11(12)-dien-5α-ol O-acetyl transacylase
A. Enzyme Purification and Library construction
Biochemical studies have indicated that the third specific intermediate of the paclitaxel biosynthesis pathway is taxa-4(20),11(12)-dien-5α-yl acetate, because this metabolite serves as a precursor of a series of polyhydroxy taxanes en route to the end-product (Hezari and Croteau, Planta Medica 63:291–295, 1997). The responsible enzyme, taxadienol acetyl transacylase, that converts taxadienol to the C5-acetate ester is, thus, an important candidate for cDNA isolation for the purpose of overexpression in relevant producing organisms to increase paclitaxel yield (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999).
This enzyme has been partially purified and characterized with respect to reaction parameters (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999); however, the published fractionation protocol does not yield a pure protein suitable for amino acid microsequencing that is required for an attempt at reverse genetic cloning of the gene. It is also important to note that the gene has no homologs or orthologs (i.e., other terpenoid or isoprenoid O-acetyl transacylases) in the databases to permit similarity-based cloning approaches.
Using methyl jasmonate-induced Taxus canadensis cells as an enriched enzyme source, a new isolation and purification protocol (see FIG. 3, and protocol described infra) was developed to efficiently yield homogeneous protein for microsequencing. Although the protein was N-blocked and failed to yield peptides that could be internally sequenced by V8 (endoproteinase Glu-C, Roche Molecular Biochemical, Nutley, N.J.) proteolysis or cyanogen bromide (CNBr) cleavage, treatment with endolysC (endoproteinase Lys-C, Roche Molecular Biochemical, Nutley, N.J.) and trypsin yielded a mixture of peptides. Five of these could be separated by high-performance liquid chromatography (HPLC) and verified by mass spectrometry (MS), and yielded sequence information useful for a cloning effort (FIG. 2).
For cDNA library construction, a stable, methyl jasmonate-inducible T. cuspidata suspension cell line was chosen for mRNA isolation because the production of paclitaxel was highly inducible in this system (which permits the preparation of a suitable subtractive library, if necessary). The mixing of experimental protocols as used with different Taxus species is not a significant limitation, since all Taxus species are known to be very closely related and are considered by several taxonomists to represent geographic variants of the basic species T. baccata (Bolsinger and Jaramillo, Silvics of Forest Trees of North America (revised), Pacific Northwest Research Station, USDA, p. 17, Portland, Oreg., 1990; and Voliotis, Isr. J. Botany. 35:47–52, 1986). Thus, the genes encoding geranylgeranyl diphosphate synthase and taxadiene synthase (early steps of paclitaxel biosynthesis) from T. canadensis and T. cuspidata evidence only very minor sequence differences. Hence, a method was developed for the isolation of high-quality mRNA from Taxus cells (Qiagen, Valencia, Calif.) and this material was employed for cDNA library construction using a commercial kit which is available from STRATAGENETM, La Jolla, Calif.
B. Reverse Genetic Cloning
Of the five peptides that were generated with proteolytic enzymes and were sequenced (FIG. 2), peptide SEQ ID NOs: 30, 31, and 33 were found to exhibit some similarity to the sequences of the only two other plant acetyl transacylases that have been documented, namely, deacetylvindoline O-acetyl transacylase involved in indole alkaloid biosynthesis (St. Pierre et al., Plant J. 14:703–713, 1998) and benzyl alcohol O-acetyl transacylase involved in the biosynthesis of aromatic esters of floral scent (Dudareva et al., Plant J. 14:297–304, 1998). Lesser resemblance was found to a putative aromatic O-benzoyl transacylase of plant origin (Yang et al., Plant Mol. Biol. 35:777–789, 1997). Of the five peptide sequences (FIG. 2), SEQ ID NO: 30 was most suitable for primer design based on codon degeneracy considerations, and two such forward degenerate primers, AT-FOR1 (SEQ ID NO: 34) and AT-FOR2 (SEQ ID NO: 35), were synthesized (FIG. 4). A search of the database with the tryptic peptide ILVYYPPFAGR (SEQ ID NO: 30) revealed two possible variants of this sequence among several gene entries of known and unknown function (these entries are listed in Table 1). Consideration of these distantly related sequences allowed the design of two additional forward degenerate primers (AT-FOR3 (SEQ ID NO: 36) and AT-FOR4 (SEQ ID NO: 37)), and permitted identification of a distal consensus sequence from which a degenerate reverse primer (AT-REV1 (SEQ ID NO: 38)) was designed (FIG. 4). (An alignment of the Taxus sequences with the extant database sequence entries of Table 1 illustrates the lack of significant homology between the Taxus sequences and any previously described genes.)







TABLE 1







Database (GENBANK TM) sequences used for peptide comparisons.


For alignment, see FIG. 6; for placement in dendrogram, see FIG. 7. The


accession number is followed by a two-letter code indicating genus and


species (AT, Arabidopsis thaliana; CM, Cucumis melo; CR,


Catharanthus roseus; DC, Dianthus caryophyllus; CB, Clarkia breweri;


NT, Nicotiana tabacum).










Protein




Identification


Accession No.
No.
Function





AC000103_AT
g2213627
unknown; from genomic sequence for


SEQ ID NO: 61

Arabidopsis thaliana BAC F21J9


AC000103_AT
g2213628
unknown; from genomic sequence for


SEQ ID NO: 62

A. thaliana BAC F21J9


AF002109_AT
g2088651
unknown; hypersensitivity-related gene


SEQ ID NO: 63

201 isolog


AC002560_AT
g2809263
unknown; from genomic sequence for


SEQ ID NO: 64

A. thaliana BAC F21B7


AC002986_AT
g3152598
unknown; similarity to C2-HC type


SEQ ID NO: 65

zinc finger protein C.e-MyT1




gb/U67079 from C. elegans and to




hypersensitivity-related gene 201 isolog




T28M21.14 from A. thaliana BAC


AC002392_AT
g3176709
putative anthranilate


SEQ ID NO: 69

N-hydroxycinnamoyl/benzoyltransferase


AL031369_AT
g3482975
unknown; putative protein


SEQ ID NO: 70


Z84383_AT
g2239083
hydroxycinnamoyl:benzoyl-CoA:


SEQ ID NO: 73

anthranilate N-hydroxycinnamoyl:




benzoyl transferase


Z97338_AT
g2244896
unknown; similar to HSR201 protein N.


SEQ ID NO: 74

tabacum


Z97338_AT
g2244897
unknown; hypothetical protein


SEQ ID NO: 75


AL049607_AT
g4584530
unknown; putative protein


SEQ ID NO: 76


AF043464_CB
g3170250
acetyl CoA:benzylalcohol acetyl


SEQ ID NO: 66

transferase


Z70521_CM
g1843440
unknown; expressed during ripening of


SEQ ID NO: 72

melon (Cucumis melo L.) fruits


AF053307_CR
g4091808
deacetylvindoline 4-O-acetyl transferase


SEQ ID NO: 68


AC004512_DC
g3335350
unknown; similar to gb/Z84386


SEQ ID NO: 67

anthranilate N-hydroxycinnamoyl/




benzoyltransferase from Dianthus




caryophyllus


X95343_NT
g1171577
unknown; hypersensitive reaction in


SEQ ID NO: 71
tobacco









PCR amplifications were performed using each combination of forward and reverse primers, and induced Taxus cell library cDNA as a target. The amplifications produced, by cloning and sequencing, twelve related but distinct amplicons (each ca. 900 bp) having origins from the various primers (Table 2). These amplicons are designated “Probe 1” through “Probe 12,” and their nucleotide and deduced amino acid sequences are listed as SEQ ID NOs: 1–24, respectively.







TABLE 2







Primer combinations, amplicons and acquired genes. The parentheses and brackets are


used to designate the primer pair used and the corresponding frequency at which that


primer pair amplified the probe.










Amplicon












Size

Acquired Gene












Primer Pair
(bp)
Frequency
Designation
Designation
Function





AT-FOR1/AT-
920
7/12
Probe 1
TAX1 (full-length)
taxadienol


REV1

(12/31)

SEQ ID NO: 27;
acetyl


(AT-FOR2/AT-



SEQ ID NO: 28
transferase


REV1)










(FIG. 4)
SEQ ID NO: 1; SEQ ID NO: 2
TAX2 (full-length)
benzoyl




SEQ ID NO: 25;
transferase




SEQ ID NO: 26












AT-FOR1/AT-
920
7/12
Probe 2
Probe 2 was not
—


REV1

(2/31)

used, but likely


(AT-FOR2/AT-



would have acquired


Rev1)



TAX2 because






the sequence






corresponds directly






to this gene.









(FIG. 4)
SEQ ID NO: 3; SEQ ID NO: 4













AT-FOR4/AT-
903
2/29
Probe 3
—
—


REV1









(FIG. 4)
SEQ ID NO: 5; SEQ ID NO: 6













AT-FOR3/AT-
908
1/29
Probe 4
—
—


REV1










(FIG. 4)
SEQ ID NO: 7; SEQ ID NO: 8
—
—












AT-FOR4/AT-
908
1/32
Probe 5
TAX5 (full-length)
transacylase


REV1



SEQ ID NO: 49;






SEQ ID NO: 50










(FIG. 4)
SEQ ID NO: 9; SEQ ID NO: 10
—
—












AT-FOR4/AT-
911
8/32
Probe 6
TAX6 (full-length)
10-deacetylbaccatin


REV1

(1/29)

SEQ ID NO: 44;
III-10-O-acetyl


(AT-FOR3/AT-

[1/32]

Seq ID No: 45
transferase


REV1)


[AT-FOR4/AT-


REV1]










(FIG. 4)
SEQ ID NO: 11;
—
—



SEQ ID NO: 12












AT-FOR3/AT-
968
6/29
Probe 7
TAX7 (full-length)
transacylase


REV1



SEQ ID NO: 51;






SEQ ID NO: 52










(FIG. 4)
SEQ ID NO: 13;
—
—



SEQ ID NO: 14












AT-FOR3/AT-
908
1/29
Probe 8
—
—


REV1

(2/32)


(AT-FOR4/AT-


REV1)










(FIG. 4)
SEQ ID NO: 15;
—
—



SEQ ID NO: 16












AT-FOR2/AT-
908
1/32
Probe 9
TAX9 (full-length)
transacylase


REV1

(5/29)

SEQ ID NO: 59,


(AT-FOR3/AT-



SEQ ID NO: 60


REV1)










(FIG. 4)
SEQ ID NO: 17;
—
—



SEQ ID NO: 18












AT-FOR4/AT-
911
2/32
Probe 10
TAX10 (full-length)
transacylase


REV1



SEQ ID NO: 53;






SEQ ID NO: 54










(FIG. 4)
SEQ ID NO: 19;
—
—



SEQ ID NO: 20












AT-FOR4/AT-
920
1/32
Probe 11
—
—


REV1










(FIG. 4)
SEQ ID NO: 21;
—
—



SEQ ID NO: 22












AT-FOR3/AT-
908
3/29
Probe 12
TAX12 (full-length)
transacylase


REV1

(1/32)

SEQ ID NO: 55,


(AT-FOR4/AT-



SEQ ID NO: 56


REV1)










(FIG. 4)
SEQ ID NO: 23;
—
—



SEQ ID NO: 24



TAX13 does not appear to
TAX13 (full-length)
transacylase



directly correspond to any of
SEQ ID NO: 57;



the above listed Probes
SEQ ID NO: 58









Notably, Probe 1, derived from the primers AT-FOR1 (SEQ ID NO: 34) and AT-REV1 (SEQ ID NO: 38), amplified a  ̃900 bp DNA fragment encoding, with near identity, the proteolytic peptides corresponding to SEQ ID NOs: 31–33 of the purified protein. These results suggested that the amplicon Probe 1 represented the target gene for taxadienol acetyl transacylase. Probe 1 was then 32P-labeled and employed as a hybridization probe in a screen of the methyl jasmonate-induced T. cuspidata suspension cell λZAP IITM cDNA library. Standard hybridization and purification procedures ultimately led to the isolation of three full-length, unique clones designated TAX1 (SEQ ID NO: 28), TAX2 (SEQ ID NO: 26), and TAX6 (SEQ ID NOS: 27, 25, and 44, respectively).
C. Sequence Analysis and Functional Expression
Clone TAX1 bears an open reading frame of 1317 nucleotides (nt; SEQ ID NO: 27)) and encodes a deduced protein of 439 amino acids (aa; SEQ ID NO: 28) with a calculated molecular weight of 49,079 kDa. Clone TAX2 bears an open reading frame of 1320 nt (SEQ ID NO: 25) and encodes a deduced protein of 440 aa (SEQ ID NO: 26) with a calculated molecular weight of 50,089 kDa. Probe 6 (SEQ ID NO: 3) was found to bear 80% nucleotide-level homology to TAX1 (SEQ ID NO: 27), suggesting that the full-length version of probe 6 may encode anotehr taxoid acetyl transferase. Probe 6 was 32P-labeled and used as a probe to screen a Taxus cell suspension cDNA library, the full-length cDNA was obtained and designated as TAX6. Clone TAX6 bears an open reading frame of 1320 nt (SEQ ID NO: 44) and encodes a deduced protein of 440 aa (SEQ ID NO: 45) with a calculated molecular weight of 49,000 kDa.
The sizes of TAX1 (SEQ ID NO: 28) and TAX2 (SEQ ID NO: 26) are consistent with the molecular weight of the native taxadienol transacetylase (MW  ̃50,000) determined by gel-permeation chromatography (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999) and SDS polyacrylamide gel electrophoresis (SDS-PAGE). The deduced amino acid sequences of both TAX1 (SEQ ID NO: 28) and TAX2 (SEQ ID NO: 26) also remotely resemble those of other acetyl transacylases (50–56% identity; 64–67% similarity) involved in different pathways of secondary metabolism in plants (St. Pierre et al., Plant J. 14:703–713, 1998; and Dudareva et al., Plant J. 14:297–304, 1998). When compared to the amino acid sequence information from the tryptic peptide fragments, TAX1 (SEQ ID NO: 28) exhibited a very close match (91% identity), whereas TAX2 (SEQ ID NO: 26) exhibited conservative differences (70% identity).
The TAX6 (SEQ ID NO: 45) calculated molecular weight of 49,052 kDa is consistent with that of the native TAX6 (SEQ ID NO: 45) protein (50 kDa), determined by gel permeation chromatography, indicating the protein to be a functional monomer, and is very similar to the size of the related, monomeric taxadien-5α-ol transacetylase (MW=49,079). The acetyl CoA:10-deacetylbacctin III-10-O-acetyl transferase from Taxus cuspidata appears to be substantially different in size from the acetyl CoA:10-hydroxytaxane-O-acetyl transferase recently isolated from Taxus chinensis and reported at a molecular weight of 71,000 (Menhard and Zenk, Phytochemistry 50:763–774, 1999).
The deduced amino acid sequence of TAX6 (SEQ ID NO: 45) resembles that of TAX1 (SEQ ID NO: 28) (64% identity; 80% similarity) and those of other acetyl transferases (56–57% identity; 65–67% similarity) involved in different pathways of secondary metabolism in plants (Dudareva et al., Plant J. 14:297–304, 1998; St-Pierre et al., Plant J. 14:703–713, 1998). Additionally both TAX6 (SEQ ID NO: 45) and TAX2 (SEQ ID NO: 26) possess the HXXXDG (SEQ ID NO: 48) (residues H162, D166, and G167 in TAX6 (SEQ ID NO: 45), and residues H159, D163, and G164 in TAX2 (SEQ ID NO: 26), respectively) motif found in other acyl transferases (Brown et al., J. Biol. Chem. 269:19157–19162, 1994; Carbini and Hersh, J. Neurochem. 61:247–253, 1993; Hendle et al., Biochemistry 34:4287–4298, 1995; and Lewendon et al., Biochemistry 33:1944–1950, 1994); this sequence element has been suggested to function in acyl group transfer from acyl CoA to the substrate alcohol (St. Pierre et al., Plant J 14:703–713, 1998).
To determine the identity of the putative taxadienol acetyl transacylase, TAX1 (SEQ ID NO: 28), TAX2 (SEQ ID NO: 26), and TAX6 (SEQ ID NO: 45) were subcloned in-frame into the expression vector pCWori+ (Barnes, Methods Enzymol. 272:3–14, 1996) and expressed in E. coli JM109 cells. The transformed bacteria were cultured and induced with isopropyl β-D-thiogalactoside (IPTG), and cell-free extracts were prepared and evaluated for taxadienol acetyl transacylase activity using the previously developed assay procedures (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999). Clone TAX1 (corresponding directly to Probe 1) expressed high levels of taxadienol acetyl transacylase activity (20% conversion of substrate to product), as determined by radiochemical analysis; the product of this recombinant enzyme was confirmed as taxadienyl-5α-yl acetate by gas chromatography-mass spectrometry (GC-MS) (FIGS. 5A–5G). Clone TAX2 did not express taxadienol acetyl transacylase activity and was inactive with the [3H]taxadienol and acetyl CoA co-substrates. However, the clone TAX2 may encode an enzyme for a step later in the paclitaxel biosynthetic pathway (TAX2 has been shown to correspond to Probe 2). Neither of the recombinant proteins expressed from TAX1 (SEQ ID NO: 28) or TAX2 (SEQ ID NO: 26) was capable of acetylating the advanced paclitaxel precursor 10-deacetyl baccatin III to baccatin III. Thus, based on the demonstration of functionally expressed activity, and the resemblance of the recombinant enzyme in substrate specificity and other physical and chemical properties to the native form, clone TAX1 was confirmed to encode the Taxus taxadienol acetyl transacylase.
Additionally, the heterologously expressed TAX6 (SEQ ID NO: 45) was partially purified by anion-exchange chromatography (O-diethylaminoethylcellulose, Whatman, Clifton, N.J.) and ultrafiltration (AMICONTM DIAFLOTM YM 10 membrane, MILLIPORETM, Bedford, Mass.) to remove interfering hydrolases from the bacterial extract, and the recombinant enzyme was determined to catalyze the conversion of 10-deacetylbaccatin III to baccatin III; the latter is the last diterpene intermediate in the paclitaxel biosynthetic pathway. The optimum pH for TAX6 (SEQ ID NO: 45) was determined to be 7.5, with half-maximal velocities at pH 6.4 and 7.8. The Km values for 10-deacetylbaccatin III and acetyl CoA were determined to be 10 μM and 8 μM, respectively, by Lineweaver-Burk analysis (for both plots R2=0.97). These kinetic constants for TAX6 (SEQ ID NO: 45) are comparable to the taxa-4(20),11(12)-dien-5α-ol acetyl transferase possessing Km values for taxadienol and acetyl CoA of 4 μM and 6 μM, respectively. The TAX6 (SEQ ID NO: 45) appears to acetylate the 10-hydroxyl group of taxoids with a high degree of regioselectivity, since the enzyme does not acetylate the 1β-, 7β-, or 13α-hydroxyl groups of 10-deacetylbaccatin III, nor does it acetylate the 5α-hydroxyl group of taxa-4(20),11(12)-dien-5α-ol.
III. Other Transacylases of the Paclitaxel Pathway
A. Overview
Screening for transacylase activity can be done by initially obtaining and/or synthesizing substrates, as described below. The individual transacylase enzymes, derived from cDNA clones TAX2, TAX5, TAX7, TAX9, TAX10, TAX12, and TAX13 (SEQ ID NOS: 25, 49, 51, 59, 53, 55, and 57, respectively) are expressed in bacteria as described below. The expressed enzymes are subjected to a crude purification procedure and screened for their ability to act upon the following substrates: 2-debenzoyl-7,13-diacetylbaccatin III, N-debenzoyltaxol, benzoyl CoA, and co-substrates, baccatin III and phenylisoserinyl CoA. The results from these screening assays can identify which of the above-mentioned clones is a debenzoyltaxol-N-benzoyl transferase and which is a baccatin III-13-O-phenylisoserinyl transferase.
B. Production of Suitable Substrates
1. 2-debenzoyl-7,13-diacetylbaccatin III
Authentic 10-deacetylbaccatin III was obtained from either Hauser Chemical Research Inc. (Boulder, Colo.), Dabur India Ltd. (New Delhi, India), or Natland International Corp. (Morrisville, N.C.). (2α,5α)-Dihydroxy-taxa-4(20),11(12)-diene was a generous gift from Robert Williams (Colorado State University, Boulder, Colo.). Methyl jasmonate was obtained from Bedoukian Research, Inc. (Danbury, Conn.). Benzoyl coenzyme A as the sodium salt was purchased from Sigma Chemical Co. (St. Louis, Mo.). [7-14C]Benzoic acid was purchased from NEN Life Sciences Products (Boston, Mass.). All other reagents were purchased from ALDRICHTM Chemical Co. (Milwaukee, Wis.), unless noted otherwise, and were used without further purification.
The synthesis of 2-debenzoyl-7,13-diacetylbaccatin III was initiated by first synthesizing 7,13-diacetylbaccatin III as follows: To a stirred solution of 10-deacetylbaccatin III (160 mg, 294 μmol) in dry CH2Cl2 (5 mL) at 25° C. under nitrogen were added acetic anhydride (20 equiv.), dimethylaminopyridine (20 equiv.), and triethylamine (50 μL, 361 μmol). After 16 hours, the reaction was diluted with EtOAc (50 mL) and quenched with water (10 mL). The mixture was stirred for 15 minutes, and the aqueous fraction was separated and extracted with EtOAc (2×25 mL). The combined organic fractions were washed with brine, 0.1 N HCl, water, and dried over anhydrous MgSO4. The organic solvent was evaporated under reduced pressure, and the crude product was purified by silica gel flash column chromatography (EtOAc:hexane, 60:40, v/v) to yield pure 7,13-diacetylbaccatin III (see FIG. 11) (180 mg, 91% yield, 99% purity by 1H-NMR). 1H-NMR (300 MHz, CDCl3)δ: 1.10 (s, CH3), 1.14 (s, CH3), 1.74 (s, CH3), 1.77 (ddd, J=1.8, 10.8, and 14.7 Hz, H-6β), 1.90 (d, J=1.2 Hz, vinyl-CH3) 1.97 (s, C(O)CH3), 2.12 (s, C(O)CH3), 2.14 (s, C(O)CH3), 2.17 (d, J=8.7 Hz, H-14), 2.29 (s, C(O)CH3), 2.53 (ddd, J=7.2, 9.6, and 14.4 Hz, H-6α), 3.89 (d, J=6.9 Hz, H-3), 4.09 (d, J=8.4 Hz, H-20α), 4.24 (d, J=8.4 Hz, H-20β), 4.91 (dd, J=1.8 and 9.6 Hz, H-5), 5.53 (dd, J=7.2 and 10.5 Hz, H-7), 5.60 (d, J=6.9 Hz, H-2), 6.10 (dt, J=1.2 and 8.7 Hz, H-13), 6.19 (s, H-10), 7.39–8.01 (aromatic protons). APCI-MS: m/z 671 (PH+).
Bis(2-methoxyethoxy)-aluminum hydride (65+ wt % in toluene, 3 equiv.) was added dropwise to a stirred solution of 7,13-diacetylbaccatin III (170 mg, 253 μmol) in dry THF (2 mL) at 0° C. After stirring for 30 minutes at 0° C., the reaction was quenched by dropwise addition of saturated NH4Cl. The mixture was stirred for 10 minutes, then warmed to room temperature and diluted with EtOAc (50 mL), followed by addition of water (10 mL). The aqueous phase was separated and extracted again with EtOAc (2×25 mL). The combined organic fractions were washed with brine and water, then dried over anhydrous MgSO4. The solvent was evaporated and the crude product was purified by silica gel flash column chromatography (40–60% EtOAc gradient in hexane) to yield 2-debenzoyl-7,13-diacetylbaccatin III (see FIG. 11) (60 mg, 42% yield, 99% purity by 1H-NMR). 1H-NMR (300 MHz, CDCl3)δ: 1.04 (s, CH3), 1.22 (s, CH3), 1.77 (s, CH3), 1.85 (ddd, J=1.8, 10.8, and 14.4 Hz, H-6β), 1.90 (d, J=1.2 Hz, vinyl-CH3), 2.02 (s, C(O)CH3), 2.14 (s, C(O)CH3), 2.15 (s, C(O)CH3), 2.20 (s, C(O)CH3), 2.58 (ddd, J=7.2, 9.6, and 14.4 Hz, H-6α), 2.65 (d, J=5.1 Hz, OH at C-2), 3.58 (d, J=6.9 Hz, H-3), 3.90 (dd, J=5.4 and 6.5 Hz, H-2), 4.49 (d, J=9.6 Hz, H-20α), 4.63 (d, J=9.6 Hz, H-20β), 4.97 (dd, J=1.8 and 9.6 Hz, H-5), 5.55 (dd, J=7.2 and 10.7 Hz, H-7), 6.15 (dt, J=1.2 and 9.6 Hz, H-13), 6.18 (s, H-10), H-14 proton signal obscured. APCI-MS: m/z 567 (PH+)
Regarding the synthesis of [7-14C]benzoyl coenzyme A, the following method was adapted from a procedure described for the synthesis of long-chain acyl CoA esters (Rasmussen et al., Biochem. J. 265:849–855, 1990). To a solution of [7-14C]benzoic acid (3.3 mg, 27 μmol, sp. ac. 18.5 Ci/mol) in CH2Cl2:THF (5:2, v/v, 1.4 mL) under nitrogen was added 1 M triethylamine in CH2Cl2 (3.0 μL, 30 μmol) in one portion. The mixture was stirred together for 10 minutes at room temperature. Ethyl chloroformate (2.57 μL, 2.9 mg, 27 μmol) was added in one portion and the reaction was stirred for 1 hour at room temperature. The solvents were evaporated, and the residue was dissolved in 0.5 mL t-butanol. Coenzyme A as the sodium salt (23 mg, 30 μmol dissolved in 0.5 mL of 0.4 M NaHCO3) was added to the butanolic solution, and the reaction mixture was stirred for 0.5 hours at room temperature. The reaction mixture was quenched with 1 M HCl (200 μL) and adjusted to pH 5 with 15 mM NaH2PO4 (pH 4.8). The solvents were evaporated in vacuo (5 hours) at room temperature. To remove residual t-butanol, the sample was dissolved in dry methanol, and the solvent removed under vacuum. The remaining residue was resuspended in 15 mM NaH2PO4 (pH 6.9, 7 mL). The crude product was purified using a C18 SEP-PAKTM cartridge (500 mg C18 silica gel, MILLIPORETM Corp, Milford, Mass.) that was first washed with methanol (2×6 mL) and water (2×6 mL), then finally equilibrated with 15 mM NaH2PO4 (pH 6.9, 2×6 mL). The entire volume of crude sample was loaded onto the column eluted with 5 mL portions of increasing methanol in 15 mM NaH2PO4 (pH 6.9) to yield [7-14C]benzoyl coenzyme A as the sodium salt (eluted in 10–15% methanol). The chemical purity (99%) of the synthetic CoA thioester was assessed by analytical TLC (silica gel developed with n-butanol:H2O:AcOH, 5:3:2, v/v/v) and comparison to authentic, co-chromatographed benzoyl coenzyme A (Rf=0.4). The TLC plate was air dried at 25° C., and the radiochemical purity of the synthetic product was determined to be 99% by liquid scintillation counting of scraped regions of the plate co-migrating with authentic standard, and regions both below and above it. The [7-14C]benzoyl CoA so isolated was lyophilized, resuspended in water (10 mL) to reconstitute to 15 mM NaH2PO4, and then carefully adjusted to pH 5 with 1 N HCl. The solution was extracted with ether (3×5 mL) to remove trace organic contaminants, and the residual ether was evaporated under a stream of nitrogen to yield a 1.35 mM solution of [7-14C]benzoyl coenzyme A.
2. Production of N-debenzoyltaxol
N-debenzoyltaxol can be prepared by adding excess units of N-acylamino acid amidohydrolase (Sigma Chemical Co., St. Louis, Mo.) from porcine kidney to a suspension of  ̃5 mg N-debenzoyl-(N-phenylacetyltaxol) (a gift from Hauser Chemical Research (Boulder, Colo.)). The resulting solution is stirred at room temperature. After 18 hours, the solution is acidified to pH 3 with 1 N HCl and extracted with ether. The aqueous fraction is lyophilized, the remaining residue can be dissolved in a minimal volume of buffer, and the crude product can be purified by cation exchange column chromatography. Purified N-debenzoyltaxol can be precipitated with a dilute solution of NH4OH, placed on ice for 0.5 hours, and centrifuged to pellet the product. The supernatant is decanted, the precipitate rinsed with a minimal volume of water, and the product lyophilized. The isolated product can be authenticated by 1H-NMR on a VARIANTM MERCURYTM 300 (with deuterated chloroform as solvent and internal reference) and direct injection mass spectrometry (HEWLETT-PACKARDTM Series 1100 MSD system in atmospheric pressure chemical ionization mode).
3. Synthesis of Phenylisoserinyl CoA
Phenylisoserinyl CoA can be prepared by dissolving N-benzoyl-(2R,3S)-3-phenylisoserine (200 mg) in 5 mL of water. The resulting solution can be acidified to pH 2 with 1 N HCl and refluxed for 5 hours. The reaction mixture is cooled to room temperature and extracted with ether, and the aqueous fraction is lyophilized to dryness. The remaining crude product can be dissolved in a minimum volume of water and adjusted to pH 5 with dilute NH4OH to recrystallize the (2R,3S)-3-phenylisoserine. The product purity can be accessed by 1H-NMR (with D2O as solvent and internal reference). Excess units of acyl-coenzyme A synthetase (Sigma Chemical Co., St. Louis, Mo.) and coenzyme A (Sigma Chemical Co., St. Louis, Mo.) are added to a solution of (2R,3S)-3-phenylisoserine (ALDRICHTM Chemical Co., Milwaukee, Wis.) dissolved in an appropriate buffer (pH 8). The product can be purified by C18 column chromatography and the product purity determined by silica gel TLC and 1H-NMR (with D2O as solvent and internal reference).
B. Crude Isolation of the Enzymes
TAX2, TAX5, TAX7, TAX9, TAX10, TAX 12, and TAX13 (SEQ ID NOS: 25, 49, 44, 60, 51, 53, 55, and 57, respectively) were expressed in bacteria using the following protocol. For a typical preparation of the recombinant enzyme, each of the E. coli cultures transformed with a pCWori+ vector harboring a putative transacylase gene was grown overnight at 37° C. in 5 mL Luria-Bertani medium supplemented with 50 μg ampicillin/mL. 1 mL of these cultures was individually added to separate aliquots of 100 mL Terrific Broth culture medium (6 g bacto-tryptone (Difco Laboratories, Spark, Md.), 12 g yeast extract (EM Science, Cherryhill, N.J.), and 2 mL glycerol in 500 mL water) and grown at 37° C. After 3 hours, the medium was supplemented with 1 mM isopropyl-β-D-thiogalactopyranoside for induction and 50 μg ampicillin/mL, and the cultures were grown at 25° C. After 18 hours, the bacteria were harvested by centrifugation, resuspended in 25 mL of assay buffer (25 mM Mopso, pH 7.4), and disrupted by sonication at 0° C. The resulting homogenates were centrifuged at 15,000×g to pellet debris. The resulting supernatants were centrifuged at 90,000×g to provide the soluble enzyme fractions.
C. Screening Assay
A 1-mL aliquot of each soluble enzyme preparation was incubated with 2-debenzoyl-7,13-diacetylbaccatin III (500 μM) and [7-14C]benzoyl coenzyme A (500 μM, 9.3 μCi) for 1.5 hours at 31° C. The reaction mixtures were extracted with ether (2 mL), and the organic phases were removed and concentrated in vacuo. The resulting crude products of the assay were dissolved in acetonitrile (50 μL) and analyzed by radio-HPLC using a Perkin Elmer HPLC ISS 200 coupled to a PACKARD RADIOMATICTM radioactivity detector series A100 (Canberra, Ind., Meriden, Conn.) (see FIGS. 12A and 12B for a representative chromatogram). The samples were separated on a VYDACTM (Hesperia, Calif.) C18 column (5μ 250×4.6 mm) by elution at 1 mL/min with a linear gradient starting from 30:70 CH3CN:H2O to 60:40 CH3CN:H2O over 30 minutes, then to 100% CH3CN with a linear gradient over 5 minutes, held at 100% CH3CN for 5 minutes, and, finally, returned to the initial conditions over 5 minutes. A detected biosynthetic product absorbing at A228 and possessing a coincidental radioactivity response with the same retention time as authentic 7,13-diacetylbaccatin III directed efforts towards large-scale preparation (4 L) of the enzyme expressed from a single E. coli JM109 transformant bearing the clone designated TAX2. The product generated by large-scale preparation of the putative TAX2 (SEQ ID NO: 26) benzoyl transferase ( ̃50μg) was analyzed by combined liquid chromatography-mass spectrometry (LC-MS) using a HEWLETT-PACKARDTM Series 1100 MSD in the atmospheric pressure chemical ionization (APCI) mode. The sample, dissolved in acetonitrile (200 μL), was loaded (5 μL) onto a PHENOMENEXTM (Torrance, Calif.) CUROSIL-GTM column (5μ 250×4.6 mm) that was eluted at 1 mL/minute with 30:70 CH3CN:H2O for 5 minutes, increased linearly to 80:20 CH3CN:H2O over 55 minutes, and then held for 5 minutes (with return to initial conditions over 0.1 minutes and equilibration for 10 minutes). The column effluent was directed to the APCI inlet of the mass spectrometer for analysis (see FIG. 13A for a representative spectrum). Additional enzymatic product ( ̃500 μg) was purified by silica gel TLC (ethyl acetate:hexane, 60:40, v/v). The band co-migrating with authentic 7,13-diacetylbaccatin III (Rf=0.33) was isolated, dissolved in 0.5 mL deuterated chloroform as internal standard, and analyzed by proton nuclear magnetic resonance spectroscopy (1H-NMR) using a VARIANTM MERCURYTM 300 instrument.
D. Kinetic Characterization Assays
Large-scale (4 L) cultures of E. coli JM109 cells harboring the recombinant Taxus TAX2 (SEQ ID NO: 26) gene were grown, harvested, and extracted as described above. Following preparation of the soluble enzyme fraction and demonstration (by sodium dodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) analysis) that a protein of the appropriate size ( ̃50 kDa) was expressed in operationally soluble form, the protein fraction (100 mL) was applied to a column of O-diethylaminoethyl-SEPHAROSETM (2.8×20 cm, Sigma Chemical Co. (St. Louis, Mo.)) that was previously washed with 25 mM Mopso buffer (pH 7.4) containing 3 mM dithiothreitol and 1 M NaCl, and then equilibrated with this buffer (without NaCl). Following removal of unbound material, protein was eluted with a linear gradient of from 0 to 200 mM NaCl in equilibration buffer (200 mL total volume, at 3 mL/minute). Fractions containing the benzoyl transferase activity (eluting at  ̃175 mM NaCl) were combined ( ̃100 mL) and subjected to ultrafiltration (AMICONTM YM 10 membrane, MILLIPORETM, Bedford, Mass.) and repeated dilution to reduce the salt concentration to  ̃5 mM. This enzyme preparation was used for kinetic evaluation of the heterologously expressed 2-debenzoyl-7,13-diacetylbaccatin III-2-O-benzoyl transferase (TAX2; SEQ ID NO: 26), and can be used to produce other transacylase enzymes such as baccatin III-13-O-phenylisoserinyl transacylase, and debenzoyltaxol-N-benzoyl transacylase.
For kinetic evaluations, linearity with respect to protein concentration and time was first established, and the concentrations of 2-debenzoyl-7,13-diacetylbaccatin III (0–1000 μM) and [7-14C]benzoyl CoA (0–1000 μM) were independently varied while the remaining reactant was maintained at saturation (2 mM). Double reciprocal plots were constructed for each data set, and the equation of the best-fit line (R2=0.99) was determined (KALEIDAGRAPHTM, version 3.08, SYNERGY SOFTWARETM, Reading, Pa.). To examine the influence of pH on benzoyl transferase activity, enzyme preparations (1 mL,  ̃80–100 μg protein) were concentrated (NANOSEPTM 30 microconcentrator, Gelman Laboratory, Ann Arbor, Mich.) to 150 μL and then diluted, respectively, with 1.35 mL Mes (pH 6), Mopso (pH 7), glycine (pH 8), Capso (pH 9), or Caps (pH 10 and 11) buffers, all at 25 mM containing 3 mM dithiothreitol, before the assay.
E. Predicted Activity
Comparison of the deduced amino acid sequences of TAX5, TAX7, TAX9, TAX10, TAX 12, and TAX13 (SEQ ID NOS: 50, 52, 60, 54, 56, and 58, respectively) with that of TAX2 (SEQ ID NO: 26), defined as a benzoyl transferase, revealed that TAX13 encodes a peptide sequence bearing the highest amino acid similarity (74%) and identity (68%) (see Table 3 below and FIGS. 14 and 15) to the Taxus cuspidata taxane-2α-O-benzoyl transferase, suggesting that TAX13 encodes another taxane benzoyl transferase.


















TABLE 3







TAX1
TAX2
TAX5
TAX6
TAX7
TAX9
TAX10
TAX12
TAX13
PCHCBT


























TAX1

S: 74%,
S: 71%,
S: 74%,
S: 74%,
S: 76%,
S: 71%,
S: 74%,
S: 72%,
S: 66%,




I: 68%
I: 62%
I: 66%
I: 66%
I: 69%
I: 64%
I: 64%
I: 63%
I: 54%


TAX2


S: 70%,
S: 70%,
S: 70%,
S: 71%,
S: 69%,
S: 71%,
S: 74%,
S: 70%,





I: 63%
I: 65%
I: 64%
I: 64%
I: 60%
I: 63%
I: 68%
I: 57%


TAX5



S: 69%,
S: 74%,
S: 70%,
S: 67%,
S: 67%,
S: 67%,
S: 64%,






I: 62%
I: 67%
I: 62%
I: 59%
I: 57%
I: 59%
I: 52%


TAX6




S: 73%,
S: 74%,
S: 74%,
S: 72%,
S: 72%,
S: 63%,







I: 66%
I: 68%
I: 66%
I: 66%
I: 63%
I: 50%


TAX7





S: 76%,
S: 73%,
S: 71%,
S: 70%,
S: 67%,








I: 68%
I: 65%
I: 61%
I: 64%
I: 55%


TAX9






S: 74%,
S: 73%,
S: 71%,
S: 65%,









I: 66%
I: 66%
I: 58%
I: 54%


TAX10







S: 69%,
S: 67%,
S: 64%,










I: 61%
I: 58%
I: 53%


TAX12








S: 68%,
S: 66%,











I: 58%
I: 53%


TAX13









S: 67%,












I: 56%









Additionally, comparison of the deduced peptide sequences of TAX2, TAX5, TAX7, TAX9, TAX10, TAX 12, and TAX13 (SEQ ID NOS: 25, 49, 44, 60, 51, 53, 55, and 57, respectively) with that of a dual-function phenylpropanoyl/benzoyl transacylase (namely, an anthranilate hydroxycinnamoyl/benzoyl tranferase (PCHCBT) isolated from Dianthus caryophyllus (Yang et al., Plant Mol. Biol. 35:777–789, 1997)), revealed the highest similarity (67%) and identity ( ̃56%) (see Table 3, FIGS. 14 and 15) between the deduced peptide of TAX7 and PCHCBT, suggesting that TAX7 encodes the C 13 phenylisoserinyl (a phenylpropanoid) transferase.
IV. Isolating a Gene Encoding acetyl CoA:taxa-4(20),11(12)-dien-5α-ol O-acetyl transacylase
A. Experimental Overview
A newly designed isolation and purification method is described below for the preparation of homogeneous taxadien-5α-ol acetyl transacylase from Taxus canadensis. The purified protein was N-terminally blocked, thereby requiring internal amino acid microsequencing of fragments generated by proteolytic digestion. Peptide fragments so generated were purified by HPLC and sequenced, and one suitable sequence was used to design a set of degenerate PCR primers. Several primer combinations were employed to amplify a series of twelve related, gene-specific DNA sequences (Probes 1–12). Nine of these gene-specific sequences were used as hybridization probes to screen an induced Taxus cuspidata cell cDNA library. This strategy allowed for the successful isolation of nine full-length transacylase cDNA clones. The identity of one of these clones was confirmed by sequence matching to the peptide fragments described above and by heterologous functional expression of transacylase activity in Escherichia coli. 
B. Culture of Cells
Initiation, propagation and induction of Taxus sp. cell cultures, reagents, procedures for the synthesis of substrates and standards, and general methods for transacylase isolation, characterization and assay have been previously described (Hefner et al., Arch. Biochem. Biophys. 360:62–75, 1998; and Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999). Since all designated Taxus species are considered to be closely related subspecies (Bolsinger and Jaramillo, Silvics of Forest Trees of North America (revised), Pacific Northwest Research Station, USDA, Portland, Oreg., 1990; and Voliotis, Isr. J. Botany 35:47–52, 1986), the Taxus cell sources were chosen for operational considerations because only minor sequence differences and/or allelic variants between proteins and genes of the various “species” were expected. Thus, Taxus canadensis cells were chosen as the source of transacetylase because they express transacetylase at high levels, and Taxus cuspidata cells were selected for cDNA library construction because they produce paclitaxel at high levels.
C. Isolation and Purification of the Enzyme
No related terpenol transacylase genes are available in the databases (see below) to permit homology-based cloning. Hence, a protein-based (reverse genetic) approach to cloning the target transacetylase was required. This reverse genetic approach required obtaining a partial amino acid sequence, generating degenerate primers, amplifying a portion of cDNA using PCR, and using the amplified fragment as a probe to detect the correct clone in a cDNA library.
Unfortunately, the previously described partial protein purification protocol, including an affinity chromatography step, did not yield pure protein for amino acid microsequencing, nor did the protocol yield protein in useful amounts, or provide a sufficiently simplified SDS-PAGE banding pattern to allow assignment of the transacetylase activity to a specific protein (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999). Furthermore, numerous variations on the affinity chromatography step, as well as the earlier anion exchange and hydrophobic interaction chromatography steps, failed to improve the specific activity of the preparations due to the instability of the enzyme upon manipulation. Also, a five-fold increase in the scale of the preparation resulted in only marginally improved recovery (generally <5% total yield accompanied by removal of >99% of total starting protein). Furthermore, because the enzyme could not be purified to homogeneity, and attempts to improve stability by the addition of polyols (sucrose, glycerol), reducing agents (Na2S2O5, ascorbate, dithiothreitol, β-mercaptoethanol), and other proteins (albumin, casein) were also not productive (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999), this approach had to be abandoned.
To overcome the problem described above, the following isolation and purification procedure was used. The purity of the taxadienol acetyl transacylase after each fractionation step was assessed by SDS-PAGE according to Laemmli (Laemmli, Nature 227:680–685, 1970); quantification of total protein after each purification step was carried out by the method of Bradford, Anal. Biochem. 72:248–254, 1976, or by Coommassie Blue staining, and transacylase activity was assessed using the methods described in Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999.
Procedures for protein staining have been described (Wray et al., Anal. Biochem. 118:197–203, 1991). The preparation of the T. canadensis cell-free extracts and all subsequent procedures were performed at 0–4° C. unless otherwise noted. Cells (40 g batches) were frozen in liquid nitrogen and thoroughly pulverized for 1.5 minutes using a mortar and pestle. The resulting frozen powder was transferred to 225 mL of ice cold 30 mM HEPES buffer (pH 7.4) containing 3 mM dithiothreitol (DTT), XAD-4 polystyrene resin (12 g) and polyvinylpolypyrrolidone (PVPP, 12 g) to adsorb low molecular weight resinous and phenolic compounds. The slurry was slowly stirred for 30 minutes, and the mixture was filtered through four layers of cheese cloth to remove solid absorbents and particulates. The filtrate was centrifuged at 7000 g for 30 minutes to remove cellular debris, then at 100,000 g for 3 hours, followed by 0.2-μm filtration to yield a soluble protein fraction (in  ̃200 mL buffer) used as the enzyme source.
The soluble enzyme fraction was subjected to ultrafiltration (DIAFLOTM YM 30 membrane, MILLIPORETM, Bedford, Mass.) to concentrate the fraction from 200 mL to 40 mL and to selectively remove proteins of molecular weight lower than the taxadien-5α-ol acetyl transacylase (previously established at 50,000 Da in Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999). Using a peristaltic pump, the concentrate (40 mL) was applied (2 mL/minute) to a column of O-diethylaminoethylcellulose (2.8×10 cm, WHATMANTM DE-52, Fairfield, N.J.) that had been equilibrated with “equilibration buffer” (30 mM HEPES buffer (pH 7.4) containing 3 mM DTT). After washing with 60 mL of equilibration buffer to remove unbound material, the proteins were eluted with a step gradient of the same buffer containing 50 mM (25 mL), 125 mM (50 mL), and 200 mM (50 mL) NaCl.
The fractions were assayed as described previously (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999), and those containing taxadien-5α-ol acetyl transacylase activity (125-mM and 200-mM fractions) were combined (100 mL,  ̃160 mM) and diluted to 5 mM NaCl (160 mL) by ultrafiltration (DIAFLOTM YM 30 membrane, MILLIPORETM, Bedford, Mass.) and repeated dilution with 30 mM HEPES buffer (pH 7.4) containing 3 mM DTT.
Further purification was effected by high-resolution anion-exchange and hydroxyapatite chromatography run on a PHARMACIATM FPLC system coupled to a 280-nm effluent detector. The preparation described above was applied to a preparative anion-exchange column (10×100 mm, Source 15Q, PHARMACIATM Biotech., Piscataway, N.J.) that was previously washed with “wash buffer” (30 mM HEPES buffer (pH 7.4) containing 3 mM DTT) and 1 M NaCl, and then equilibrated with wash buffer (without NaCl). After removing unbound material, the applied protein was eluted with a linear gradient of 0 to 200 mM NaCl in equilibration buffer (215 mL total volume; 3 mL/minute) (see FIG. 3A). Fractions containing transacetylase activity (eluting at  ̃80 mM NaCl) were combined and diluted to 5 mM NaCl by ultrafiltration using 30 mM HEPES buffer (pH 7.4) containing 3 mM DTT as diluent, as described above. The desalted protein sample (70 mL) was loaded onto an analytical anion-exchange column (5×50 mm, Source 15Q, PHARMACIATM Biotech., Piscataway, N.J.) that was washed and equilibrated as before. The column was developed using a shallow, linear salt gradient with elution to 200 mM NaCl (275 mL total volume, 1.5 mL/minute, 3.0 mL fractions). The taxadienol acetyl transacylase eluted at  ̃55–60 mM NaCl (see FIG. 3B), and the appropriate fractions were combined (15 mL), reconstituted to 45 mL in 30 mM HEPES buffer (pH 6.9) and applied to a ceramic hydroxyapatite column (10×100 mm, BIO-RADTM Laboratories, Hercules, Calif.) that was previously washed with 200 mM sodium phosphate buffer (pH 6.9) and then equilibrated with an “equilibration buffer” (30 mM HEPES buffer (pH 6.9) containing 3 mM DTT (without sodium phosphate)). The equilibration buffer was used to desorb weakly associated material, and the bound protein was eluted by a gradient from 0 to 40 mM sodium phosphate in equilibration buffer (125 mL total volume, at 3.0 mL/minute, 3.0 mL fractions) (see FIG. 3C). The fractions containing the highest activity, eluting over 27 mL at 10 mM sodium phosphate, were combined and shown by SDS-PAGE to yield a protein of  ̃95% purity (a minor contaminant was present at  ̃35 kDa, see FIG. 3D). The level of transacylase activity was measured after each step in the isolation and purification protocol described above. The level of activity recovered is shown in Table 4.







TABLE 4







Summary of taxadien-5α-ol O-acetyl


transferase purification from Taxus cells.












Total
Total 
Specific Activity




activity
Protein
(pkat/mg
Purification



(pkat)
(mg)
protein)
(fold)















Crude extract
302
1230
0.25
1


YM30 ultrafiltration
136
98
1.4
5.6


DE-52
122
69
1.8
7.2


YM30 ultrafiltration
54
55
1.0
4


Source 15Q
47
3
16
63


(10 × 100 mm)


YM30 ultrafiltration
19
2.6
7.3
29


Source 15Q
13
0.12
108
400


(5 × 50 mm)


Hydroxyapatite
10
0.05
200
800









D. Amino Acid Microsequencing of Taxadienol Acetyl Transacylase
The purified protein from multiple preparations as described above (>95% pure,  ̃100 pmol, 50 μg) was subjected to preparative SDS-PAGE (Laemmli, Nature 227:680–685, 1970). The protein band at 50 kDa, corresponding to the taxadienol acetyl transacylase, was excised. Whereas treatment with V8 protease or treatment with cyanogen bromide (CNBr) failed to yield sequencable peptides, in situ proteolysis with endolysC (Caltech Sequence/Structure Analysis Facility, Pasadena, Calif.) and trypsin (Fernandez et al., Anal. Biochem. 218:112–118, 1994) yielded a number of peptides, as determined by HPLC, and several of these were separated, verified by mass spectrometry (Fernandez et al., Electrophoresis 19:1036–1045, 1998), and subjected to Edman degradative sequencing, from which five distinct and unique amino acid sequences (designated SEQ ID NOs: 29–33) were obtained (FIG. 2).
E. cDNA Library construction and Related Manipulations
A cDNA library was constructed from mRNA isolated from T. cuspidata suspension culture cells that had been induced to maximal paclitaxel production with methyl jasmonate for 16 hours. An optimized protocol for the isolation of total RNA from T. cuspidata cells was developed empirically using a buffer containing 100 mM Tri-HCl (pH 7.5), 4 M guanidine thiocyanate, 25 mM EDTA and 14 mM β-mercaptoethanol. Cells (1.5 g) were disrupted at 0–4° C. using a POLYTRONTM ultrasonicator (Kinematica AG, Switzerland; 4×15 second bursts at power setting 7), the resulting homogenate was adjusted to 2% (v/v) TRITONTM X-100 and allowed to stand 15 minutes on ice. An equal volume of 3 M sodium acetate (pH 6.0) was then added, and the mixed solution was incubated on ice for an additional 15 minutes, followed by centrifugation at 15,000 g for 30 minutes at 4° C. The resulting supernatant was mixed with 0.8 volume of isopropanol and allowed to stand on ice for 5 minutes, followed by centrifugation at 15,000 g for 30 minutes at 4° C. The resulting pellet was dissolved in 8 mL of 20 mM Tris-HCl (pH 8.0) containing 1 mM EDTA, adjusted to pH 7.0 by addition of 2 mL of 2 M NaCl in 250 mM MOPS buffer (pH 7.0), and total RNA was recovered by passing this solution over a nucleic acid isolation column (QIAGENTM, Valencia, Calif.) following the manufacturer's instructions. Poly(A)+ mRNA was then purified from total RNA by chromatography on oligo(dT) beads (OLIGOTEXTM mRNA Kit, QIAGENTM), and this material was used to construct a library using the λZAPIITM cDNA synthesis kit and GIGAPACKTM III gold packaging kit from STRATAGENETM, La Jolla, Calif., by following the manufacturer's instructions.
Unless otherwise stated, standard methods were used for DNA manipulations and cloning (Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and for PCR amplification procedures (Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, 1990). DNA was sequenced using AMPLITAQTM (Hoffmann-La Roche INC., Nutley, N.J.) DNA polymerase and cycle sequencing (fluorescence sequencing) on an ABI PRISMTM 373 DNA Sequencer. The E. coli strains XL1-BLUETM and XL1-BLUE MRF′TM (STRATAGENETM, La Jolla, Calif.) were used for routine cloning of PCR products and for cDNA library construction, respectively. E. coli XL1-BLUE MRF′TM cells were used for in vivo excision of purified pBLUESCRIPTTM SK from positive plaques and the excised plasmids were used to transform E. coli SOLR cells.
F. Degenerate Primer Design and PCR Amplification
Due to codon degeneracy, only one sequence of the five tryptic peptide fragments obtained (SEQ ID NO: 30 of FIG. 2) was suitable for PCR primer construction. Two such degenerate forward primers, designated AT-FOR1 (SEQ ID NO: 34) and AT-FOR2 (SEQ ID NO: 35), were designed based on this sequence (FIG. 4). Using the NCBI BLASTTM 2.0 database searching program (Genetics computer Group, Program Manual for the Wisconsin Package, version 9, Genetics computer Group, 575 Science Drive, Madison, Wis., 1994) to search for this sequence element among the few defined transacylases of plant origin (St. Pierre et al., Plant J. 14:703–713, 1998; Dudareva et al., Plant J. 14:297–304, 1998; and Yang et al., Plant Mol. Biol. 35:777–789, 1997), and the many deposited sequences of unknown function, allowed the identification of two possible sequence variants of this element (FYPFAGR (SEQ ID NO: 39) and YYPLAGR (SEQ ID NO: 40)) from which two additional degenerate forward primers, designated AT-FOR3 (SEQ ID NO: 36) and AT-FOR4 (SEQ ID NO: 37), were designed (FIG. 4). The sequences employed for this comparison are listed in Table 1. Using this range of functionally defined and undefined sequences, conserved regions were sought for the purpose of designing a degenerate reverse primer (the distinct lack of similarity of the Taxus sequences to genes in the database can be appreciated by reference to FIG. 6), from which one such consensus sequence element (DFGWGKP) (SEQ ID NO: 41) was noted, and was employed for the design of the reverse primer AT-REV1 (SEQ ID NO: 38) (FIG. 4). This set of four forward primers and one reverse primer incorporated a varied number of inosines, and ranged from 72- to 216-fold degeneracy. The remaining four proteolytic peptide fragment sequences (SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 of FIG. 2) were not only less suitable for primer design, but they were not found (by NCBI BLASTTM searching) to be similar to other related sequences, thus suggesting that these represented more specific sequence elements of the Taxus transacetylase gene.
Each forward primer (150 μM) and the reverse primer (150 μM) were used in separate PCR reactions performed with Taq polymerase (3 U/100 μL reaction containing 2 mM MgCl2) and employing the induced T. cuspidata cell cDNA library (108 PFU) as template under the following conditions: 94° C. for 5 minutes, 32 cycles at 94° C. for 1 minute, 40° C. for 1 minute and 74° C. for 2 minutes and, finally, 74° C. for 5 minutes. The resulting amplicons (regions amplified by the various primer combinations) were analyzed by agarose gel electrophoresis (Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and the products were extracted from the gel, ligated into pCR TOPOT7 (INVITROGENTM, Carlsbad, Calif.), and transformed into E. coli TOPIOF′ cells (INVITROGENTM, Carlsbad, Calif.). Plasmid DNA was prepared from individual transformants and the inserts were fully sequenced.
The combination of primers AT-FOR1 (SEQ ID NO: 34) and AT-REV1 (SEQ ID NO: 38) yielded a 900-bp amplicon. Cloning and sequencing of the amplicon revealed two unique sequences designated “Probe 1” (SEQ ID NO: 1) and “Probe 2” (SEQ ID NO: 3) (Table 2). The results with the remaining primer combinations are provided in Table 2.
G. Library Screening
Four separate library-screening experiments were designed using various combinations of the radio-labeled amplicons (Probes 1–12, described supra) as probes. Use of radio-labeled Probe 1 (SEQ ID NO: 1), led to the identification of TAX1 (SEQ ID NO: 27) and TAX2 (SEQ ID NO: 25), and use of radio-labeled Probe 6 (SEQ ID NO: 11) led to the identification of TAX6 (SEQ ID NO: 44). A probe consisting of a mixture of radio-labeled Probe 10 (SEQ ID NO: 19) and Probe 12 (SEQ ID NO: 23) led to the identification of TAX10 (SEQ ID NO: 44) and TAX12 (SEQ ID NO: 55). Finally, a probe containing a mixture of radio-labeled Probes 3, 4, 5, 7, and 9 led to the identification of TAX5, TAX7, TAX9, and TAX13 (SEQ ID NOs. 49, 51, 59, and 57, respectively). Details of these individual library-screening experiments are provided below.
The identification of TAX1 (SEQ ID NO: 27) and TAX2 (SEQ ID NO: 25) was accomplished using 1 μg of Probe 1 (SEQ ID NO: 1) that had been amplified by PCR, the resulting amplicon was gel-purified, randomly labeled with [α-32P]CTP (Feinberg and Vogelstein, Anal. Biochem. 137:216–217, 1984), and used as a hybridization probe to screen membrane lifts of 5×105 plaques grown in E. coli XL1-BLUE MRF′TM. Phage DNA was cross-linked to the nylon membranes by autoclaving on fast cycle 3–4 minutes at 120° C. After cooling, the membranes were washed 5 minutes in 2×SSC, then 5 minutes in 6×SSC (containing 0.5% SDS, 5× Denhardt's reagent, 0.5 g Ficoll (Type 400, PHARMACIATM, Piscataway, N.J.), 0.5 g polyvinylpyrrolidone (PVP-10), and 0.5 g bovine serum albumin (Fraction V, Sigma, Saint Louis, Mo.) in 100 mL total volume). Hybridization was then performed for 20 hours at 68° C. in 6×SSC, 0.5% SDS and 5× Denhardt's reagent. The nylon membranes were then washed two times for 5 minutes in 2×SSC with 0.1% SDS at 25° C., and then washed 2×30 minutes with 1×SSC and 0.1% SDS at 68° C. After washing, the membranes were exposed for 17 hours to KODAKTM (Rochester, N.Y.) XAR film at −70° C. (Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Of the plaques exhibiting positive signals ( ̃600 total), 60 were purified through two additional rounds of hybridization. Purified λZAPTMII clones were excised in vivo as pBLUESCRIPTTM II SK(−) phagemids and transformed into E. coli SOLR cells (STRATAGENETM, La Jolla, Calif.). The size of each cDNA insert was determined by PCR using T3 and T7 promoter primers, and size-selected inserts (>1.5 kb) were partially sequenced from both ends to sort into unique sequence types and to acquire full-length versions of each (by further screening with a newly designed 5′-probe, if necessary).
The same basic screening protocol, as illustrated by the results provided below, can be repeated with all of the probes described in Table 2, with the goal of acquiring the full range of full-length, in-frame putative transacylase clones for test of function by expression in E. coli. In the case of Probe 1 (SEQ ID NO: 1), two unique full-length clones, designated TAX1 (SEQ ID NO: 27 and SEQ ID NO: 28) and TAX2 (SEQ ID NO: 25 and SEQ ID NO: 26), were isolated.
An additional transacylase, TAX6 (SEQ ID NO: 44), was identified by using 40 ng of radio-labeled Probe 6 (SEQ ID NO: 11) to screen the T. cuspidata library. This full-length clone was 99% identical to Probe 6 (SEQ ID NO: 11) and its deduced amino acid sequence was 99% identical to that of Probe 6 (SEQ ID NO: 12), indicating that the probe had located its cognate.
Using 40 ng of radio-labeled Probe 10 (SEQ ID NO: 19) and 40 ng of radio-labeled Probe 12 (SEQ ID NO: 23) led to the identification of the full-length transacylases TAX10 (SEQ ID NO: 53 and SEQ ID NO: 54) and TAX12 (SEQ ID NO: 55 and SEQ ID NO: 56) in separate hybridization screening experiments.
Use of a probe mixture containing about 6 ng each of Probes 3, 4, 5, 7, and 8 (SEQ ID NOs. 5, 7, 9, 13, and 15, respectively) randomly labeled with [α-32P]CTP (Feinberg and Vogelstein, Anal Biochem. 137:216–217, 1984) resulted in the identification of full-length transacylases TAX5 (SEQ ID NO: 49), TAX7 (SEQ ID NO: 51), and TAX9 (SEQ ID NO: 50), which correspond to Probes 5 (SEQ ID NO: 9), 7 (SEQ ID NO: 13), and 9 (SEQ ID NO: 17), respectively. An additional full-length transacylase, TAX13 (SEQ ID NO: 57) was also identified, however, this transacylase does not correspond to any of the Probes identified in Table 2.
H. cDNA Expression in E. coli 
Full-length insert fragments of the relevant plasmids are excised and subcloned in-frame into the expression vector pCWori+ (Barnes, Methods Enzymol. 272:3–14, 1996). This procedure may involve the elimination of internal restriction sites and the addition of appropriate 5′- and 3′-restriction sites for directional ligation into the expression vector using standard PCR protocols (Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990) or commercial kits such as the QUIKCHANGETM Mutagenesis System (STRATAGENETM, La Jolla, Calif.). For example, the full-length transacylase corresponding to probe 6 (SEQ ID NO: 11) was obtained using the primer set (5′-GGGAATTCCATATGGCAGGCTCAACAGAATTTGTGG-3′ (SEQ ID NO: 46) and 3′-GTTTATACATTGATTCGGAACTAGATCTGATC-5′ (SEQ ID NO: 47)) to amplify the putative full-length acetyl transferase gene and incorporate NdeI and XbaI restriction sites at the 5′- and 3′-termini, respectively, for directional ligation into vector pCWori+ (Barnes, Methods Enzymol. 272:3–14, 1996). All recombinant pCWori+ plasmids are confirmed by sequencing to insure that no errors have been introduced by the polymerase reactions, and are then transformed into E. coli JM109 by standard methods.
Isolated transformants for each full-length insert are grown to A600=0.5 at 37° C. in 50 mL Luria-Bertani medium supplemented with 50 μg ampicillin/mL, and a 1-mL inoculum added to a large scale (100 mL) culture of Terrific Broth (6 g bacto-tryptone, DIFCOTM Laboratories, Spark, Md., 12 g yeast extract, EM Science, Cherryhill, N.J., and 2 mL glycerol in 500 mL water) containing 50 μg ampicillin/mL and thiamine HCl (320 mM) and grown at 28° C. for 24 hours. Approximately 24 hours after induction with 1 mM isopropyl β-D-thiogalactoside (IPTG), the bacterial cells are harvested by centrifugation, disrupted by sonication in assay buffer consisting of 30 mM potassium phosphate (pH 7.4), or 25 mM MOPSO (pH 7.4), followed by centrifugation to yield a soluble enzyme preparation that can be assayed for transacylase activity.
I. Enzyme Assay
A specific assay for acetyl CoA:taxa-4(20),11(12)-dien-5α-ol O-acetyl transacylase has been described previously (Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999, herein incorporated by reference). Generally the assay for taxoid acyltransacylases involves the CoA-dependent acyl transfer from acetyl CoA (or other acyl or aroyl CoA ester) to a taxane alcohol, and the isolation and chromatographic separation of the product ester for confirmation of structure by GC-MS (or HPLC-MS) analysis. For another example of such an assay, see Menhard and Zenk, Phytochemistry 50:763–774, 1999.
The activity of TAX6 (SEQ ID NO: 45) was assayed under standard conditions described in Walker et al., Arch. Biochem. Biophys. 364:273–279, 1999, with 10-deacetylbaccatin III (400 μM, Hauser Chemical Research Inc., Boulder, Colo.) and [2-3H]acetyl CoA (0.45 μCi, 400μM (NEN, Boston, Mass.)) as co-substrates. The TAX6 (SEQ ID NO: 45) enzyme preparation yielded a single product from reversed-phase radio-HPLC analysis, with a retention time of 7.0 minutes (coincident radio and UV traces) corresponding exactly to that of authentic baccatin III (generously provided by Dr. David Bailey of Hauser Chemical Research Inc., Boulder, Colo.) (FIGS. 9A and 9B). The identity of the biosynthetic product was further verified as baccatin III by combined LC-MS (liquid chromatography-mass spectrometry) analysis (FIGS. 10A and 10B), which demonstrated the identical retention time (8.6×0.1 minute) and mass spectrum for the product and authentic standard. Finally, a sample of the biosynthetic product, purified by silica gel analytical TLC, produced a 1H-NMR spectrum identical to that of authentic baccatin III, confirming the enzyme as 10-deacetylbaccatin III-10-O-acetyl transferase (TAX6 (SEQ ID NO: 45)) and also confirming that the corresponding gene had been isolated.
EXAMPLES
1. Transacylase Protein and Nucleic acid Sequences
As described above, the invention provides transacylases and transacylase-specific nucleic acid sequences. With the provision herein of these transacylase sequences, the polymerase chain reaction (PCR) may now be utilized as a preferred method for identifying and producing nucleic acid sequences encoding the transacylases. For example, PCR amplification of the transacylase 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. Transacylase 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 Laboratory Manual 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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 transacylases may be amplified using primers directed towards regions of homology between the 5′ and 3′ ends of the TAX1 and TAX2 sequences. Example primers for such a reaction are:

    
    
        primer 1: 5′CCT CAT CTT TCC CCC ATT GAT AAT 3′ (SEQ ID NO: 42)
        primer 2: 5′ AAA AAG AAA ATA ATT TTG CCA TGC AAG 3′ (SEQ ID NO: 43).
    
    


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 of PCR products obtained by these amplification procedures is recommended to facilitate confirmation of the amplified sequence and to provide information on natural variation between transacylase sequences. Oligonucleotides derived from the transacylase sequence may be used in such sequencing methods.
Oligonucleotides that are derived from the transacylase 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 transacylase sequences. To enhance amplification specificity, oligonucleotide primers comprising at least 15, 20, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of these sequences may also be used.
A. Transacylases in Other Plant Species
Orthologs of the transacylase genes are present in a number of other members of the Taxus genus. With the provision herein of the transacylase nucleic acid sequences, the cloning by standard methods of cDNAs and genes that encode transacylase orthologs in these other species is now enabled. As described above, orthologs of the disclosed transacylase genes have transacylase 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 transacylase sequences using the NCBI BLASTTM 2.0 (gapped blastp set to default parameters). Proteins with even greater similarity 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 transacylase orthologs. Common to both of these techniques is the hybridization of probes or primers that are derived from the transacylase 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 any of various plant species, or RT-PCR may be performed using mRNA extracted from plant cells using standard methods. PCR primers will comprise at least 10 consecutive nucleotides of the transacylase sequences. One of skill in the art will appreciate that sequence differences between the transacylase 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. Where 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 transacylase 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 then purified and the cloned sequence contained in that colony or plaque is isolated and characterized.
Orthologs of the transacylases alternatively may be obtained by immunoscreening of an expression library. With the provision herein of the disclosed transacylase nucleic acid sequences, the enzymes may be expressed and purified in a heterologous expression system (e.g., E. coli) and used to raise antibodies (monoclonal or polyclonal) specific for transacylases. Antibodies may also be raised against synthetic peptides derived from the transacylase 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 Spring Harbor Press, Cold Spring Harbor, N.Y. 1988. Such antibodies can then be used to screen an expression cDNA library produced from a plant. This screening will identify the transacylase ortholog. The selected cDNAs can be confirmed by sequencing and enzyme activity assays.
B. Paclitaxel Transacylase Variants
With the provision of the transacylase amino acid sequences (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 45, 50, 52, 54, 56, and 58) and the corresponding cDNA (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 44, 49, 51, 53, 55, and 57), variants of these sequences can now be created.
Variant transacylases include proteins that differ in amino acid sequence from the transacylase sequences disclosed, but that retain transacylase biological activity. Such proteins may be produced by manipulating the nucleotide sequence encoding the transacylase 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 5 shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions.










TABLE 5







Original
Conservative



Residue
Substitutions









ala
ser



arg
lys



asn
gln; his



asp
glu



cys
ser



gln
asn



glu
asp



gly
pro



his
asn; gln



ile
leu; val



leu
ile; val



lys
arg; gln; 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, i.e., selecting residues that differ more significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which: (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. The effects of these amino acid substitutions or deletions or additions may be assessed for transacylase derivatives by analyzing the ability of the derivative proteins to catalyse the conversion of one paclitaxel precursor to another paclitaxel precursor.
Variant transacylase cDNA or genes may be produced by standard DNA mutagenesis techniques, for example, M13 primer mutagenesis. Details of these techniques are provided in Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., vols. 1–3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, Ch. 15. By the use of such techniques, variants may be created that differ in minor ways from the transacylase cDNA or gene sequences, yet that still encode a protein having transacylase 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 transacylase 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 transacylase amino acid sequences. For example, the fifteenth amino acid residue of the TAX2 (SEQ ID NO: 26) is alanine. This is encoded in the open reading frame (ORF) by the nucleotide codon triplet GCG. Because of the degeneracy of the genetic code, three other nucleotide codon triplets—GCA, GCC, and GCT—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 transacylase protein but that vary from the disclosed nucleic acid sequences by virtue of the degeneracy of the genetic code.
Variants of the transacylase may also be defined in terms of their sequence identity with the transacylase amino acid and nucleic acid sequences described supra. As described above, transacylases have transacylase biological activity and share at least 60% sequence identity with the disclosed transacylase sequences. Nucleic acid sequences that encode such proteins may readily be determined simply by applying the genetic code to the amino acid sequence of the transacylase, and such nucleic acid molecules may be readily produced by assembling oligonucleotides corresponding to portions of the sequence.
As previously mentioned, another method of identifying variants of the transacylases is nucleic acid hybridization. Nucleic acid molecules that are derived from the transacylase cDNA and gene sequences include molecules that hybridize under various conditions to the disclosed paclitaxel transacylase nucleic acid molecules, or fragments thereof. 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.












Very High Stringency (detects sequences


that share 90% sequence identity)











Hybridization in
5×
SSC at
65° C.
16 hours


Wash twice in
2×
SSC at
room temp.
15 minutes each


Wash twice in
0.5×
SSC at
65° C.
20 minutes each







High Stringency (detects sequences that


share 80% sequence identity or greater)











Hybridization in
5×
SSC at
65° C.
16 hours


Wash twice in
2×
SSC at
room temp.
20 minutes each


Wash once in
1×
SSC at
55° C.
30 minutes each







Low Stringency (detects sequences that


share greater than 50% sequence identity)











Hybridization in
6×
SSC at
room temp.
16 hours


Wash twice in
3×
SSC at
room temp.
20 minutes each





(20–21° C.)









The sequences encoding the transacylases identified through hybridization may be incorporated into transformation vectors and introduced into host cells to produce transacylase.
2. Introduction of Transacylases 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 (e.g., a promoter) directing expression of the cDNA in plant cells. The transformation vector is then introduced into plant cells by any of various techniques (e.g., 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.
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 manifested 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. Pat. No. 5,571,706 (“Plant Virus Resistance Gene and Methods”)
U.S. Pat. No. 5,677,175 (“Plant Pathogen Induced Proteins”)
U.S. Pat. No. 5,510,471 (“Chimeric Gene for the Transformation of Plants”)
U.S. Pat. No. 5,750,386 (“Pathogen-Resistant Transgenic Plants”)
U.S. Pat. No. 5,597,945 (“Plants Genetically Enhanced for Disease Resistance”)
U.S. Pat. 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. Pat. No. 5,750,871 (“Transformation and Foreign Gene Expression in Brassica Species”)
U.S. Pat. No. 5,268,526 (“Overexpression of Phytochrome in Transgenic Plants”)
U.S. Pat. No. 5,262,316 (“Genetically Transformed Pepper Plants and Methods for their Production”)
U.S. Pat. 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-express the introduced cDNA. In light of the foregoing and the provision herein of the transacylase 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 transacylase activity. Furthermore, the expression of one or more transacylases in plants may give rise to plants having increased production of paclitaxel 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, plant-transformation vectors include one or more cloned plant genes (or cDNAs) under the transcriptional control of 5′- and 3′-regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter regulatory region (e.g., 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, e.g., 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 Benfey 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 paclitaxel (Han et al., Plant Science 95:187–196, 1994). Therefore, it is likely that incorporation of one or more of the described transacylases under the influence of a strong promoter (like CaMV promoter) would increase production yields of paclitaxel 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: (a) 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); (b) light (e.g., 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); (c) hormones, such as abscisic acid (Marcotte et al., Plant Cell 1:969, 1989); (d) wounding (e.g., wunl, Siebertz et al., Plant Cell 1:961, 1989); and (e) chemicals such as methyl jasmonate or salicylic acid (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; Schemthaner 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 transacylase gene promoters may be utilized. With the provision herein of the transacylase 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 will also 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 readily be ascertained by operably linking the selected sequence region to a transacylase cDNA (in conjunction with suitable 3′ regulatory region, such as the NOS 3′ regulatory region as discussed below) and determining whether the transacylase is expressed.
Plant-transformation vectors may also include RNA-processing signals, for example, introns, that may be positioned upstream or downstream of the ORF sequence in the transgene. In addition, the expression vectors may also 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 transacylase gene 3′-regulatory sequence may also be employed.
Finally, as noted above, plant-transformation vectors may also include dominant selectable marker genes to allow for the ready selection of transformants. Such genes include those encoding antibiotic-resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) and herbicide-resistance genes (e.g., phosphinothricin acetyltransacylase).
B. Arrangement of Paclitaxel Transacylase Sequence in a Vector
The particular arrangement of the transacylase sequence in the transformation vector is selected according to the type of expression of the sequence that is desired.
In most instances, enhanced transacylase activity is desired, and the transacylase ORF is operably linked to a constitutive high-level promoter such as the CaMV 35S promoter. As noted above, enhanced transacylase activity may also be achieved by introducing into a plant a transformation vector containing a variant form of the transacylase cDNA or gene, for example a form that varies from the exact nucleotide sequence of the transacylase ORF, but that encodes a protein retaining transacylase 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; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile 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 paclitaxel and related compounds.
3. Production of Recombinant Paclitaxel transacylase in Heterologous Expression Systems
Various yeast strains and yeast-derived vectors are commonly used for the expression of heterologous proteins. For instance, Pichia pastoris expression systems, obtained from INVITROGENTM (Carlsbad, Calif.), 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) (INVITROGENTM Product Catalogue, 1998, INVITROGENTM, Carlsbad Calif.).
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 well-known baculovirus system, the ecdysone-inducible expression system that uses 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 INVITROGENTM, Carlsbad, Calif.
The cloned expression vector encoding one or more transacylases 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 (ATCCTM deposit #7940), WISH cells (ATCCTM deposit #CCL-25), Daudi cells (ATCCTM deposit #CCL-213), Mandin-Darby bovine kidney cells (ATCCTM deposit #CCL-22) and Chinese hamster ovary (CHO) cells (ATCCTM deposit #CRL-2092). Common yeast cells include Pichia pastoris (ATCCTM deposit #201178) and Saccharomyces cerevisiae (ATCCTM deposit #46024). Insect cells include cells from Drosophila melanogaster (ATCCTM deposit #CRL-10191), the cotton bollworm (ATCCTM deposit #CRL-9281), and Trichoplusia ni egg cell homoflagellates. Fish cells that may be used include those from rainbow trout (ATCCTM deposit #CLL-55), salmon (ATCCTM deposit #CRL-1681), and zebrafish (ATCCTM deposit #CRL-2147). Amphibian cells that may be used include those of the bullfrog, Rana castebelana (ATCCTM deposit #CLL-41). Reptile cells that may be used include those from Russell's viper (ATCCTM deposit #CCL-140). Plant cells that could be used include Chlamydomonas cells (ATCCTM deposit #30485), Arabidopsis cells (ATCCTM deposit #54069) and tomato plant cells (ATCCTM deposit #54003). Many of these cell types are commonly used and are available from the ATCCTM as well as from commercial suppliers such as PHARMACIATM (Uppsala, Sweden), and INVITROGENTM.
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 then be characterized for activity and stability and may be used to practice any of the various methods according to the invention.
4. Creation of Transacylase-Specific Binding Agents
Antibodies to the transacylase enzymes, and fragments thereof, of the present invention may be useful for purification of the enzymes. The provision of the transacylase sequences allows for the production of specific antibody-based binding agents to these enzymes.
Monoclonal or polyclonal antibodies may be produced to the transacylases, portions of the transacylases, or variants thereof. Optimally, antibodies raised against epitopes on these antigens will specifically detect the enzyme. That is, antibodies raised against the transacylases would recognize and bind the transacylases, 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, N.Y., 1989.
To determine that a given antibody preparation (such as a preparation produced in a mouse against TAX1 (SEQ ID NO: 28)) specifically detects the transacylase by Western blotting, total cellular protein is extracted from cells and electrophoresed on an SDS-polyacrylamide gel. The proteins are then 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 non-specifically 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 a transacylase will, by this technique, be shown to bind substantially only the transacylase band (having a position on the gel determined by the molecular weight of the transacylase). 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 non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific anti-transacylase binding.
Antibodies that specifically bind to transacylases belong to a class of molecules that are referred to herein as “specific binding agents.” Specific binding agents that are capable of specifically binding to the transacylase 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 transacylase 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 AMICONTM filter device, to the level of a few micrograms per milliliter. Alternatively, peptide fragments of a transacylase may be utilized as immunogens. Such fragments may be chemically synthesized using standard methods, or may be obtained by cleavage of the whole transacylase 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 transacylase 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 transacylase amino acid sequences for producing antibodies. Thus, for example, peptides that comprise at least 10, 15, 20, 25, or 30 consecutive amino acid residues of the amino acid sequence may be employed. Monoclonal or polyclonal antibodies to the intact transacylase or peptide fragments thereof may be prepared as described below.
A. Monoclonal Antibody Production by Hybridoma Fusion
Monoclonal antibody to any of various epitopes of the transacylase 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 then 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, 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.
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., Chapter 19, in Wier (ed.), Handbook of Experimental Immunology, Blackwell, 1973. A plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/mL of serum (about 12 μM). 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 the transacylases 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 β-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. Pat. No. 5,620,896 (“DNA Vaccines Against Rotavirus Infections”); U.S. Pat. No. 5,643,578 (“Immunization by Inoculation of DNA Transcription Unit”); and U.S. Pat. No. 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. Pat. No. 5,648,237 (“Expression of Functional Antibody Fragments”); U.S. Pat. No. 4,946,778 (“Single Polypeptide Chain Binding Molecules”); and U.S. Pat. No. 5,455,030 (“Immunotherapy Using Single Chain Polypeptide Binding Molecules”), and references cited therein.
5. Paclitaxel Production In Vivo
The creation of recombinant vectors and transgenic organisms expressing the vectors are important for controlling the production of transacylases. These vectors can be used to decrease transacylase production, or to increase transacylase production. A decrease in transacylase production will likely result from the inclusion of an antisense sequence or a catalytic nucleic acid sequence that targets the transacylase encoding nucleic acid sequence. Conversely, increased production of transacylase can be achieved by including at least one additional transacylase encoding sequence in the vector. These vectors can then be introduced into a host cell, thereby altering transacylase production. In the case of increased production, the resulting transacylase may be used in in vitro systems, as well as in vivo for increased production of paclitaxel, other taxoids, intermediates of the paclitaxel biosynthetic pathway, and other products.
Increased production of paclitaxel 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 transacylases. Furthermore, the heterologous or homologous transacylase sequences can be placed under the control of a constitutive promoter, or an inducible promoter. This will lead to the increased production of transacylase, thus eliminating any rate-limiting effect on paclitaxel production caused by the expression and/or activity level of the transacylase.
6. Paclitaxel Production In Vitro
Currently, paclitaxel is produced by a semisynthetic method described in Hezari and Croteau, Planta Medica 63:291–295, 1997. This method involves extracting 10-deacetyl-baccatin III, or baccatin III, intermediates in the paclitaxel biosynthetic pathway, and then finishing the production of paclitaxel using in vitro techniques. As more enzymes are identified in the paclitaxel biosynthetic pathway, it may become possible to completely synthesize paclitaxel in vitro, or at least increase the number of steps that can be performed in vitro. Hence, the transacylases of the present invention may be used to facilitate the production of paclitaxel 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 paclitaxel, but also transgenic organisms that produce increased levels of important intermediates, such as 10-deacetyl-baccatin III and baccatin III.
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.

## Claims

1. A purified protein, comprising the amino acid sequence as set forth in SEQ ID NO: 26.

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

3. The isolated nucleic acid molecule of claim 2, comprising the nucleic acid sequence as set forth in SEQ ID No: 25.

4. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to the nucleic acid molecule of claim 2.

5. An isolated cell transformed with the recombinant nucleic acid molecule of claim 4.

6. An isolated nucleic acid molecule that:
(a) hybridizes under very high-stringency conditions with a nucleic acid probe, the probe comprising the nucleic acid sequence as set forth in SEQ ID NO: 25;and
(b) encodes a protein having taxoid transacylase activity, wherein the hybridization step is performed for 20 hours at 68° C. in 6×SSC, 0.5% SDS and 5× Denhardt's reagent, washed two times for 5 minutes in 2×SSC and 0.1% SDS at 25° C., and washed 2× for 30 minutes with 1×SSC and 0.1% SDS at 68° C.

7. An isolated transacylase encoded by the nucleic acid molecule of claim 6.

8. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to the nucleic acid molecule of claim 6.

9. An isolated cell transformed with the recombinant nucleic acid molecule of claim 8.

10. An isolated nucleic acid molecule comprising a nucleic acid sequence that:
(a) has at least 95% sequence identity with SEQ ID NO: 25;and
(b) encodes a protein having taxoid transacylase activity.

11. A purified protein having taxoid transacylase activity, comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence as set forth in SEQ ID NO: 26; and
(b) an amino acid sequence having at least 90% sequence identity to the sequence specified in (a).

12. An isolated nucleic acid molecule encoding the protein of claim 11.

13. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to the nucleic acid molecule of claim 12.

14. An isolated cell transformed with the recombinant nucleic acid molecule of claim 13.

15. A recombinant nucleic acid molecule, comprising a promoter sequence operably linked to the nucleic acid molecule of claim 10.

16. An isolated cell transformed with the recombinant nucleic acid molecule of claim 10.

17. The purified protein of claim 11, wherein the amino acid sequence has at least 95% sequence identity to SEQ ID NO: 26.