Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-05-25
2004-11-16
Achutamurthy, Ponnathapura (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S320100, C435S252300, C435S254110, C435S419000, C435S325000
Reexamination Certificate
active
06818755
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to transacylase enzymes and methods of using such enzymes to produce Taxol™ and related taxoids.
INTRODUCTION
The complex diterpenoid Taxol™ (paclitaxel) (Wani et al.,
J. Am. Chem. Soc
. 93: 2325-2327, 1971) is a potent antimitotic agent with excellent activity against a wide range of cancers, including ovarian and breast cancer (Arbuck and Blaylock,
Taxol™: Science and Applications
, CRC Press, Boca Raton, 397-415, 1995; Holmes et al.,
ACS Symposium Series
583: 31-57, 1995). Taxol™ was originally isolated from the bark of the Pacific yew (
Taxus brevifolia
). For a number of years, Taxol™ was obtained exclusively from yew bark, but low yields of this compound from the natural source coupled to the destructive nature of the harvest, prompted new methods of Taxol™ production to be developed. Taxol™ is currently produced primarily by semisynthesis from advanced taxane metabolites (Holton et al.,
Taxol™: Science and Applications
, CRC Press, Boca Raton, 97-121, 1995) that are present in the needles (a renewable resource) of various
Taxus
species. However, because of the increasing demand for this drug (both for use earlier in the course of cancer intervention and for new therapeutic applications) (Goldspiel,
Pharmacotherapy
17: 110S-125S, 1997), availability and cost remain important issues. Total chemical synthesis of Taxol™ 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 Taxol depends upon a detailed understanding of the biosynthetic pathway, the enzymes catalyzing the sequence of reactions, especially the rate-limiting steps, and the genes encoding these proteins. Isolation of genes encoding enzymes involved in the pathway is a particularly important goal, since overexpression of these genes in a producing organism can be expected to markedly improve yields of the drug.
The Taxol™ biosynthetic pathway is considered to involve more than 12 distinct steps (Floss and Mocek,
Taxol: Science and Applications
, CRC Press, Boca Raton, 191-208,1995; and Croteau et al.,
Curr. Top. Plant Physiol
. 15: 94-104, 1996), however, very few of the enzymatic reactions and intermediates of this complex pathway have been defined. The first committed enzyme of the Taxol™ pathway is taxadiene synthase (Koepp et al.,
J. Biol. Chem
. 270: 8686-8690, 1995) that cyclizes the common precursor geranylgeranyl diphosphate (Hefner et al.,
Arch. Biochem. Biophys
. 360: 62-74, 1998) to taxadiene (FIG.
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,
Taxol™: 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 Taxol™ biosynthesis is an oxygenation reaction catalyzed by taxadiene-5&agr;-hydroxylase (FIG.
1
). The enzyme, characterized as a cytoebrome 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-5&agr;-ol (Hefuer et al.,
Chem. Biol
. 3:479-489, 1996).
The third specific step of Taxol™ biosynthesis appears to be the acetylation of taxa-4(20),11 (12)-dien-5&agr;-ol to taxa-4(20),11 (12)-dien-5&agr;-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 Taxol™ 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 K
m
values of 4.2 &mgr;M and 5.5 &mgr;M for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced Taxol™ 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-5&agr;-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-5&agr;-ol O-acetyl transacylase that catalyzes the first acylation step of Taxol™ biosynthesis and genes encoding other acyl transfer steps would represent an important advance in efforts to increase Taxol™ 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 Taxol™ and related taxoids, as well as intermediates within the Taxol™ 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 Taxol™ 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 tra
Croteau Rodney B.
Schoendorf Anne
Walker Kevin D.
Wildung Mark R.
Achutamurthy Ponnathapura
Kerr Kathleen
Klarquist & Sparkman, LLP
Washington State University Research Foundation
LandOfFree
Nucleic acid molecules encoding 10-deacetylbaccatin... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid molecules encoding 10-deacetylbaccatin..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid molecules encoding 10-deacetylbaccatin... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3360429