Recombinant materials and methods for production of limonene...

Details Recombinant materials and methods for production of limonene... Recombinant materials and methods for production of limonene...

1999-04-14

2001-02-27

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S252300, C435S410000, C435S320100, C435S468000, C435S476000, C536S023200

Reexamination Certificate

active

06194185

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to nucleic acid sequences which code for cytochrome P450 limonene hydroxylases, such as (−)-limonene-6-hydroxylase from
Mentha spicata
and (−)-limonene-3-hydroxylase from
Mentha piperita
, and to vectors containing the sequences, host cells containing the sequences and methods of producing recombinant limonene hydroxylases and their mutants.
BACKGROUND OF THE INVENTION
Several hundred naturally occurring, monoterpenes are known, and essentially all are biosynthesized from geranyl pyrophosphate, the ubiquitous C
10
intermediate of the isoprenoid pathway (Croteau and Cane,
Methods of Enzymology
110:383-405 [1985]; Croteau,
Chem. Rev.
87:929-954 [1987]). Monoterpene synthases, often referred to as “cyclases,” catalyze the reactions by which geranyl pyrophosphate is cyclized to the various monoterpene carbon skeletons. Many of the resulting carbon skeletons undergo subsequent oxygenation by cytochrome P450 hydroxylases to give rise to large families of derivatives. Research on biosynthesis has been stimulated by the commercial significance of the essential oils (Guenther,
The Essential Oils
, Vols. III-VI (reprinted) R. E. Krieger, Huntington, N.Y. [1972]) and aromatic resins (Zinkel and Russell,
Naval Stores: Production, Chemistry, Utilization
, Pulp Chemicals Association, New York [1989]) and by the ecological roles of these terpenoid secretions, especially in plant defense (Gershenzon and Croteau, in “Herbivores: Their Interactions with Secondary Plant Metabolites,” Vol. I, 2nd Ed. (Rosenthal and Berenbaum, eds.) Academic Press, San Diego, Calif., pp. 165-219 [1991]; Harborne, in “Ecological Chemistry and Biochemistry of Plant Terpenoids,” (Harborne and Tomas-Barberan eds.) Clarendon Press, Oxford, Mass., pp. 399-426 [1991]).
The reactions catalyzed by the cytochrome P450-(−)-limonene hydroxylases determine the oxidation pattern of the monoterpenes derived from limonene (see FIGS.
1
A-
1
C). These reactions are completely regiospecific and are highly selective for (−)-limonene as substrate. The primary products of limonene hydroxylation (trans-carveol and trans-isopiperitenol) are important essential oil components and serve as precursors of numerous other monoterpenes of flavor or aroma significance (see FIGS.
1
A-
1
C).
One of the major classes of plant monoterpenes is the monocyclic p-menthane (1 -methyl-4-isopropylcyclohexane) type, found in abundance in members of the mint (Mentha) family. The biosynthesis of p-menthane monoterpenes in Mentha species, including the characteristic components of the essential oil of peppermint (i.e., (−)-menthol) and the essential oil of spearmint (i.e., (−)-carvone), proceeds from geranyl pyrophosphate via the cyclic olefin (−)-limonene and is followed by a series of enzymatic redox reactions that are initiated by cytochrome P450 limonene hydroxylases (e.g., limonene-3-hydroxylase in peppermint and limonene-6-hydroxylase in spearmint and related species; Karp et al.,
Arch. Biochem. Biophys.
276:219-226 [1990]; Gershenzon et al.,
Rec. Adv. Phytochem.
28:193-229 [1994]; Lupien et al.,
Drug Metab. Drug Interact.
12:245-260 [1995]). The products of limonene hydroxylation and their subsequent metabolites (as shown in
FIGS. 1A-1C
) also serve ecological roles in plant defense mechanisms against herbivores and pathogens, and may act as signals in other plant-insect relationships (e.g., as attractants for pollinators and seed dispersers).
A detailed understanding of the control of monoterpene biosynthesis and of the reaction mechanisms, enzymes and the relevant cDNA clones as tools for evaluating patterns of developmental and environmental regulation, for examining active site structure-function relationships and for the generation of transgenic organisms bearing such genes are disclosed in part in parent U.S. related application Ser. No. 08/582,802 filed Jan. 4, 1996 as a continuation of application Ser. No. 08/145,941 filed Oct. 28, 1993, the disclosures of which are incorporated herein by this reference, which disclose the isolation and sequencing of cDNAs encoding (−)4S-limonene synthase, the enzyme responsible for cyclizing geranyl pyrophosphate to obtain (−)-limonene. To date, however, no information has been available in the art regarding the protein and nucleotide sequences relating to the enzymes through which (−)-limonene is hydroxylated (by the action of (−)-limonene-6-hydroxylase to form trans-carveol or by the action of (−)-limonene-3-hydroxylase to form trans-isopiperitenol as shown in FIG.
1
).
SUMMARY OF THE INVENTION
In accordance with the foregoing, cDNAs encoding (−)-limonene hydroxylase, particularly (−)-limonene-6-hydroxylase from spearmint and (−)-limonene-3-hydroxylase from peppermint, have been isolated and sequenced, and the corresponding amino acid sequences have been deduced. Accordingly, the present invention relates to isolated nucleic acid sequences which code for the expression of limonene hydroxylase, such as the sequence designated SEQ ID No:1 which encodes (−)-limonene-6-hydroxylase (SEQ ID No:2) from spearmint (
Mentha spicata
), or the sequence designated SEQ ID No:3 which encodes (−)-limonene-3-hydroxylase (SEQ ID No:4) from peppermint (
Mentha piperita
), or the sequence designated SEQ ID No:5 which encodes another (−)-limonene-3-hydroxylase (SEQ ID No:6) from peppermint (
Mentha piperita
). In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence, e.g., a DNA sequence, which codes for limonene hydroxylases or for a base sequence sufficiently complementary to at least a portion of the limonene hydroxylase DNA or RNA to enable hybridization therewith (e.g., antisense limonene hydroxylase RNA or fragments of complementary limonene hydroxylase DNA which are useful as polymerase chain reaction primers or as probes for limonene hydroxylases or related genes). In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention. Thus, the present invention provides for the recombinant expression of limonene hydroxylases, and the inventive concepts may be used to facilitate the production, isolation and purification of significant quantities of recombinant limonene hydroxylase (or of the primary enzyme products, trans-carveol in the case of (−)-limonene-6-hydroxylase or trans-isopiperitenol in the case of (−)-limonene-3-hydroxylase) for subsequent use, to obtain expression, or enhanced expression, or altered expression, of limonene hydroxylase in plants to attain enhanced trans-carveol or trans-isopiperitenol production as a predator or pathogen defense mechanism, attractant or environmental signal, or may be otherwise employed in an environment where the regulation or expression of limonene hydroxylase is desired for the production of limonene hydroxylase or the enzyme products, trans-carveol or trans-isopiperitenol, or their derivatives.
In another aspect, the present invention is directed to isolated nucleic acid molecules that hybridize under stringent hybridization conditions to a fragment (having a length of at least 15 bases) of any one of the nucleic acid molecules of the present invention encoding a limonene-3-hydroxylase or limonene-6-hydroxylase.


REFERENCES:
patent: 93/22441 (1993-11-01), None
Alberts et al.,Molecular Biology of the Cell, Second Ed., Garland Publishing, Inc., New York, NY, pp. 185-187 and 265-266 (1989).
Alonso et al., “Production and Characterization of Polyclonal Antibodies in Rabbits to 4S-Limonene Synthase from Spearmint (Mentha spicata),”Arch. Biochem. Biophys.301(1):58-63 (1993).
Alonso et al., “Purification of 4S-Limonene Synthase, a Monoterpene Cyclase from the Glandular Trichomes of Peppermi

Affiliated with

Also associated with

Rating

Recombinant materials and methods for production of limonene... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Recombinant materials and methods for production of limonene..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant materials and methods for production of limonene... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2591436