Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing a carotene nucleus
Reexamination Certificate
1997-09-25
2003-02-25
Achutamurthy, Ponnathupura (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing a carotene nucleus
C435S233000
Reexamination Certificate
active
06524811
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention describes the DNA sequence for eukaryotic genes encoding &egr; cyclase, isopentenyl pyrophosphate isomerase (IPP) and &bgr;-carotene hydroxylase as well as vectors containing the same and hosts transformed with said vectors. The present invention also provides a method for augmenting the accumulation of carotenoids and production of novel and rare carotenoids. The present invention provides methods for controlling the ratio of various carotenoids in a host. Additionally, the present invention provides a method for screening for eukaryotic genes encoding enzymes of carotenoid biosynthesis and metabolism.
2. Discussion of the Background
Carotenoid pigments with cyclic endgroups are essential components of the photosynthetic apparatus in oxygenic photosynthetic organisms (e.g., cyanobacteria, algae and plants; Goodwin, 1980). The symmetrical bicyclic yellow carotenoid pigment &bgr;-carotene (or, in rare cases, the asymmetrical bicyclic &agr;-carotene) is intimately associated with the photosynthetic reaction centers and plays a vital role in protecting against potentially lethal photooxidative damage (Koyama, 1991). &bgr;-carotene and other carotenoids derived from it or from &agr;-carotene also serve as light-harvesting pigments (Siefermann-Harms, 1987), are involved in the thermal dissipation of excess light energy captured by the light-harvesting antenna (Demmig-Adams & Adams, 1992), provide substrate for the biosynthesis of the plant growth regulator abscisic acid (Rock & Zeevaart, 1991; Parry & Horgan, 1992), and are precursors of vitamin A in human and animal diets (Krinsky, 1987). Plants also exploit carotenoids as coloring agents in flowers and fruits to attract pollinators and agents of seed dispersal (Goodwin, 1980). The color provided by carotenoids is also of agronomic value in a number of important crops. Carotenoids are currently harvested from plants for use as pigments in food and feed.
The probable pathway for formation of cyclic carotenoids in plants, algae and cyanobacteria is illustrated in FIG.
1
. Two types of cyclic endgroups are commonly found in higher plant carotenoids, these are referred to as the &bgr; and &egr; cyclic endgroups (
FIG. 3.
; the acyclic endgroup is referred to as the &PSgr; or psi endgroup). These cyclic endgroups differ only in the position of the double bond in the ring. Carotenoids with two &bgr; rings are ubiquitous, and those with one &bgr; and one &egr; ring are common, but carotenoids with two &egr; rings are rarely detected. &bgr;-Carotene (
FIG. 1
) has two &bgr; endgroups and is a symmetrical compound that is the precursor of a number of other important plant carotenoids such as zeaxanthin and violaxanthin (FIG.
2
).
Carotenoid enzymes have previously been isolated from a variety of sources including bacteria (Armstrong et al., 1989, Mol. Gen. Genet. 216, 254-268; Misawa et al., 1990, J. Bacteriol., 172, 6704-12), fungi (Schmidhauser et al., 1990, Mol. Cell. Biol. 10, 5064-70), cyanobacteria (Chamovitz et al., 1990, Z. Naturforsch, 45c, 482-86) and higher plants (Bartley et al., Proc. Natl. Acad. Sci USA 88, 6532-36; Martinez-Ferez & Vioque, 1992, Plant Mol. Biol. 18, 981-83). Many of the isolated enzymes show a great diversity in function and inhibitory properties between sources. For example, phytoene desaturases from Synechococcus and higher plants carry out a two-step desaturation to yield &zgr;-carotene as a reaction product; whereas the same enzyme from Erwinia introduces four double bonds forming lycopene. Similarity of the amino acid sequences are very low for bacterial versus plant enzymes. Therefore, even with a gene in hand from one source, it is difficult to screen for a gene with similar function in another source. In particular, the sequence similarity between prokaryotic and eukaryotic genes is quite low.
Further, the mechanism of gene expression in prokaryotes and eukaryotes appears to differ sufficiently such that one can not expect that an isolated eukaryotic gene will be properly expressed in a prokaryotic host.
The difficulties in isolating related genes is exemplified by recent efforts to isolated the enzyme which catalyzes the formation of &bgr;-carotene from the acyclic precursor lycopene. Although this enzyme had been isolated in a prokaryote, it had not been isolated from any photosynthetic organism nor had the corresponding genes been identified and sequenced or the cofactor requirements established. The isolation and characterization of the enzyme catalyzing formation of &bgr;-carotene in the cyanobacterium Synechococcus PCC7942 was described by the present inventors and others (Cunningham et al., 1993 and 1994).
The need remains for the isolation of eukaryotic genes involved in the carotenoid biosynthetic pathway, including a gene encoding an &egr; cyclase, IPP isomerase and &bgr;-carotene hydroxylase. There remains a need for methods to enhance the production of carotenoids. There also remains a need in the art for methods for screening for eukaryotic genes encoding enzymes of carotenoid biosynthesis and metabolism.
SUMMARY OF THE INVENTION
Accordingly, a first object of this invention is to provide isolated eukaryotic genes which encode enzymes involved in carotenoid biosynthesis; in particular, &egr; cyclase, IPP isomerase and &bgr;-carotene hydroxylase.
A second object of this invention is to provide eukaryotic genes which encode enzymes which produce novel carotenoids.
A third object of the present invention is to provide vectors containing said genes.
A fourth object of the present invention is to provide hosts transformed with said vectors.
Another object of the present invention is to provide hosts which accumulates novel or rare carotenoids or which overexpress known carotenoids.
Another object of the present invention is to provide hosts with inhibited carotenoid production.
Another object of this invention is to secure the expression of eukaryotic carotenoid-related genes in a recombinant prokaryotic host.
A final object of the present invention is to provide a method for screening for eukaryotic genes which encode enzymes involved in carotenoid biosynthesis and metabolism.
These and other objects of the present invention have been realized by the present inventors as described below.
REFERENCES:
patent: 98/28545 (1996-09-01), None
Albrecht et al. Light-Stimulated Carotenoid Biosynthesis during Transformation of Maize Etioplasts Is Regulated by Increased Activity of Isopentenyl Pyrophosphate Isomerase. Plant Physiol. (1994) 105:529-534.*
Christensen et al. Enzymatic Synthesis of Isotopically Labeled Isoprenoid Diphosphates. Bioorganic & Medicinal Chemistry (1994) 2(7):631-637.*
LaGarde et al. Increased Production of Zeaxanthin and Other Pigments by Application of Genetic Engineering Techniques to Synechocystic sp. Strain PCC 6803. Applied and Environmetal Microbiology (2000) 66(1):64-72, Jan. 2000.*
Armstrong, Journal of Bacteriology, 1994, vol. 176, pp. 4795-4802.*
Street et al., Biochemistry, 1990, vol. 29, pp. 7531-7538.*
Campbell et al., Plant Mol. Biol. 36:323-328, 1997.*
Blanc et al., Plant Physiol. 111:652, Jun. 1996.*
Anderson et al., J. Biol. Chem. 264:19169-19175, Nov. 1989.*
Hahn et al., J. Biol. Chem. 270:11298-11303, May 1995.*
Chem. Abstr. 125:294752, 1996.
Cunningham, Jr. Francis X.
Sun Zairen
Achutamurthy Ponnathupura
Arent Fox Kintner Plotkin & Kahn
Kerr Kathleen
University of Maryland
LandOfFree
Methods of increasing or decreasing carotenoids and other... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods of increasing or decreasing carotenoids and other..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods of increasing or decreasing carotenoids and other... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3156217